Methods of modulating β cell function

ABSTRACT

Methods of modulating pancreatic function by modulating MCH signaling in a β cell.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/355,645, filed Jan. 31, 2003, which claims the benefit of U.S.Provisional Application No. 60/353,752, filed Jan. 31, 2002. Thecontents of both applications are incorporated herein by reference.

GOVERNMENT RIGHTS

This invention was made with U.S. government support under grant numbersDK 56113, 56116, and 09825 awarded by the National Institutes of Health.The government has certain rights in the invention.

BACKGROUND

Melanocyte concentrating hormone (MCH) is a cyclic 19 amino-acid peptidethat is an important regulator of feeding behavior. In the brain, MCH issynthesized in neurons of the lateral hypothalamus, which makemonosynaptic connections throughout the cortex. MCH neurons also synapsewith neurons in the parabrachial nucleus and the nucleus of the tractussolitarius, hindbrain nuclei important in ingestive behavior.

MCH has been shown to circulate in plasma and to stimulate leptinsecretion from rat adipocytes. When administered ICV, MCH induces anacute increase in feeding. Mice in which the MCH gene has been ablatedare hypophagic and lean.

SUMMARY OF THE INVENTION

The invention is based, in part, on the inventors' discovery that MCHmodulates pancreatic endocrine function, e.g., β-cell function, e.g.,insulin secretion. While not being bound by theory, MCH is thought tostimulate insulin release from islet β-cells directly via the MCHreceptor. In addition, MCH is believed to act as a growth factor forislet cells.

Accordingly, the invention features a method of modulating pancreaticfunction, e.g., β cell function, e.g., insulin secretion or β cell sizeor growth. The method includes modulating MCH signaling in a β cell. Ina preferred embodiment, the method includes administering to a β cell anagent that modulates, e.g., increases or decreases, MCH signaling in theβ cell. Increasing MCH signaling can lead to increased insulinsecretion, while inhibiting MCH signaling can lead to decreased insulinsecretion.

In one embodiment, the agent promotes, increases or mimics MCH signalingin a β cell, to thereby increase insulin release from the β cell, orincrease β cell size or growth. In one embodiment, the agent promotes,increases or mimics MCH signaling by binding to a protein on the surfaceof the β cell, e.g., MCH receptor, e.g., MCH-R1 or MCH-R2, and, e.g.,agonizes or mimics MCH binding. An agent that promotes, increases ormimics MCH signaling can be one or more of: an MCH peptide or afunctional analog thereof (e.g., [Ala-14]-MCH); an MCH receptor (MCH-R)polypeptide or functional variant or analog thereof; a peptide orprotein agonist of MCH-R, e.g., a protein or peptide that activates theMCH receptor; a small molecule that increases expression of MCH orMCH-R, e.g., by binding to the promoter region of the MCH or MCH-R gene;an antibody, e.g., an antibody or antigen binding fragment thereof thatbinds to MCH or MCH-R and, e.g., activates MCH-R or stabilizes thebinding of MCH to MCH-R or of MCH-R to a secondary messenger, e.g., a Gprotein, e.g., G_(i); or a nucleotide sequence encoding an MCH or MCH-Rpolypeptide or functional fragment or analog thereof. The nucleotidesequence can be a genomic sequence or a cDNA sequence. The nucleotidesequence can include: an MCH or MCH-R coding region; a promotersequence, e.g., a promoter sequence from an MCH or MCH-R gene or fromanother gene; an enhancer sequence; untranslated regulatory sequences,e.g., a 5′ untranslated region (UTR), e.g., a 5′UTR from an MCH or MCH-Rgene or from another gene, a 3′ UTR, e.g., a 3′UTR from an MCH or MCH-Rgene or from another gene; a polyadenylation site; an insulatorsequence. In another preferred embodiment, the level of an MCH or MCH-Rprotein is increased by increasing the level of expression of anendogenous MCH or MCH-R gene, e.g., by increasing transcription of theMCH or MCH-R gene or increasing MCH or MCH-R mRNA stability. In apreferred embodiment, transcription of the MCH or MCH-R gene isincreased by: altering the regulatory sequence of the endogenous MCH orMCH-R gene, e.g., by the addition of a positive regulatory element (suchas an enhancer or a DNA-binding site for a transcriptional activator);the deletion of a negative regulatory element (such as a DNA-bindingsite for a transcriptional repressor) and/or replacement of theendogenous regulatory sequence, or elements therein, with that ofanother gene, thereby allowing the coding region of the MCH or MCH-Rgene to be transcribed more efficiently.

In a preferred embodiment, an agent that promotes, increases or mimicsMCH signaling is an MCH agonist selected from: MCH and analogs thereof,including Arg-cyclo(S-S)(Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys (SEQ IDNO:2)) (Bednarek et al. (2001) Biochemistry 40(31):9379-86));pro-MCH(131-165) peptide known as neuropeptide-glutamic acid-isoleucine(NEI)-MCH (Maulon-Feraille (2002) J Pharmacol Exp Ther 302(2):766-73;Ac-REIGDEESAKFPIGRRDFDMLRCMLGRVYRPCWQV (SEQ ID NO:6));Ac-dArg(6)-cyclo(S-S)(Cys(7)-Met(8)-Leu(9)-Asn(10)-Arg(11)-Val(12)-Tyr(13)-Arg(14)-Pro(15)-Cys(16))-NH(2)(SEQ ID NO:3) (Bednarek et al. (2002) J Biol Chem 277(16):13821-6); andcompound S36057 (Audinot et al. (2001) Br J Pharmacol 133(3):371-8;Y-ADO-RCMLGRVFRPCW (SEQ ID NO:7; ADO is 8-amino-3,6-dioxyoctanol). Otheragonists are known or can be readily identified using routinetechniques.

In another embodiment, the agent decreases or inhibits MCH signaling, tothereby decrease insulin secretion or decrease islet cell growth. In oneembodiment, the agent decreases or inhibits MCH signaling by binding toa protein on the surface of the β cell, e.g., MCH receptor, e.g., MCH-R1or MCH-R2, and, e.g., inhibits MCH binding. An agent that decreases orinhibits MCH signaling can be one more of: an MCH or MCH-R antagonist(e.g., a [D-Arg¹¹]-MCH); a soluble MCH binding protein, e.g., a solubleMCH binding protein that binds to MCH and inhibits MCH binding to MCH-Ror MCH-R binding to a secondary messenger, e.g., a G protein, e.g.,G_(i); a soluble MCH-R binding protein, e.g., a soluble MCH-R bindingprotein that binds to MCH-R and inhibits MCH-R binding to MCH; anantibody or antigen binding fragment thereof that specifically binds toMCH or MCH-R, e.g., an antibody that disrupts MCH binding to MCH-R; amutated inactive MCH-R (e.g., an Asp-123-substituted MCH-R mutant) that,e.g., does not bind to MCH, or binds to MCH but disrupts anintracellular receptor signaling activity; an MCH or MCH-R nucleic acidmolecule that can bind to a cellular MCH or MCH-R nucleic acid sequence,e.g., mRNA, and inhibit expression of the protein, e.g., an antisensemolecule or MCH or MCH-R ribozyme; an agent which decreases MCH or MCH-Rgene expression, e.g., a small molecule which binds the promoter of MCHor MCH-R and decreases MCH or MCH-R gene expression. In anotherpreferred embodiment, MCH or MCH-R is inhibited by decreasing the levelof expression of an endogenous MCH or MCH-R gene, e.g., by decreasingtranscription of the MCH or MCH-R gene. In a preferred embodiment,transcription of the MCH or MCH-R gene can be decreased by: altering theregulatory sequences of the endogenous MCH or MCH-R gene, e.g., by theaddition of a negative regulatory sequence (such as a DNA-biding sitefor a transcriptional repressor), or by the removal of a positiveregulatory sequence (such as an enhancer or a DNA-binding site for atranscriptional activator).

In a preferred embodiment, an agent that decreases or inhibits MCHsignaling is an MCH antagonist selected from: SNAP-7941 (Borowsky et al.(2002) Nat Med 8(8):825-30); Leu(9)-Gly(10) and Arg(14)-Pro(15) peptideanalogs (Bednarek et al. (2002) Biochemistry 41(20):6383-90); T-226296(Takekawa et al. (2002) Eur J Pharmacol 438(3):129-35); MCH analogssubstituted in MCH-(6-17) in 6 out of 12 amino acids with concomitantreplacement of the disulfide bond by an amide bond (Audinot et al.(2001) J Biol Chem 276(17):13554-62); [D-Arg(11)]-MCH (Macdonald et al.(2000) Mol Pharmacol 58(1):217-25); and amide derivatives of1,4-di-substituted piperidine antagonists (U.S. Pat. No. 6,472,394).Other antagonists are known or can be readily identified using routinetechniques.

In a preferred embodiment, the MCH-R is MCH-R1.

In a preferred embodiment, the MCH-R is MCH-R2.

In a preferred embodiment, the agent is administered to the cell invitro, e.g., the agent is administered to a cultured β cell. In someembodiments, the cell can subsequently be implanted into a subject.Preferred cells used in this method are autologous cells. Allogenic orxenogenic cells can also be used.

In a preferred embodiment, the agent is administered ex-vivo, e.g., theagent is administered to an isolated pancreatic tissue, e.g., an isletor islet equivalent.

In a preferred embodiment, the agent is administered in-vivo, e.g., theagent is administered to a subject. In one embodiment, the animal is anexperimental animal, e.g., a rodent model for an insulin relateddisorder, e.g., a NOD Mouse and its related strains, BB Rat, Leptin orLeptin Receptor mutant rodents, Zucker Diabetic Fatty (ZDF) Rat,Sprague-Dawley rats, Obese Spontaneously Hypertensive Rat (SHROB,Koletsky Rat), Wistar Fatty Rat, New Zealand Obese Mouse, NSY Mouse,Goto-Kakizaki Rat, OLETF Rat, JCR:LA-cp Rat, NeonatallyStreptozotocin-Induced (n-STZ) Diabetic Rats, Rhesus Monkey, Psammomysobesus (fat sand rat), C57B1/6J Mouse. In another embodiment, thesubject is a human.

In a preferred embodiment, the subject is at risk for, or has, aninsulin-related disorder, e.g., diabetes, e.g., type 1 or type 2diabetes; obesity; insulin resistance; hyperinsulinemia; hypoglycemia.

In another aspect, the invention features a method of modulatingpancreatic function, e.g., islet function, e.g., insulin secretion, in asubject. The method includes: providing a pancreatic cell, e.g., anislet cell, e.g., a β cell; administering to the cell an agent thatmodulates MCH signaling, e.g., an agent described herein; and implantingthe cell into the subject. Preferred cells used in this method are cellsautologous to the subject. Allogenic or xenogenic cells can also beused.

In a preferred embodiment, the agent is a nucleic acid that encodes anMCH-R, e.g., MCH-R1, MCH-R2, SLT or its rodent equivalent.

In another aspect, the invention features a method of treating asubject, e.g., treating an insulin related disorder, e.g., diabetes,e.g., type 1 or type 2 diabetes; obesity; insulin resistance;hyperinsulinemia; hypoglycemia. The method includes (a) optionally,identifying a subject having or at risk for an insulin related disorder,e.g., an insulin related disorder described herein; and (b) modulatingMCH signaling to thereby treat the subject. Modulating MCH signalingmodulates insulin secretion in the subject. In preferred embodiments,the method includes administering to the subject an agent that modulatesMCH signaling.

In one embodiment, the agent promotes, increases or mimics MCH signalingin a β cell, to thereby increase insulin release from the β cell. In oneembodiment, the agent promotes, increases or mimics MCH signaling bybinding to a protein on the surface of the β cell, e.g., MCH receptor,e.g., MCH-R1 or MCH-R2, and, e.g., agonizes or mimics MCH binding. Anagent that promotes, increases or mimics MCH signaling can be one ormore of: an MCH peptide or a functional fragment or analog thereof(e.g., [Ala-14]-MCH); an MCH receptor (MCH-R) polypeptide or functionalvariant or analog thereof; a peptide or protein agonist of MCH-R, e.g.,a protein or peptide that activates MCH-R signaling to increase insulinsecretion activity, of MCH; a small molecule that increases expressionof MCH or MCH-R, e.g., by binding to the promoter region of the MCH orMCH-R gene; an antibody, e.g., an antibody or antigen binding fragmentthereof that binds to and stabilizes or assists the binding of MCH toMCH-R or of MCH-R to a secondary messenger, e.g., a G protein, e.g.,G_(i) or G_(o); or a nucleotide sequence encoding an MCH or MCH-Rpolypeptide or functional fragment or analog thereof. The nucleotidesequence can be a genomic sequence or a cDNA sequence. The nucleotidesequence can include: an MCH or MCH-R coding region; a promotersequence, e.g., a promoter sequence from an MCH or MCH-R gene or fromanother gene; an enhancer sequence; untranslated regulatory sequences,e.g., a 5′ untranslated region (UTR), e.g., a 5′UTR from an MCH or MCH-Rgene or from another gene, a 3′ UTR, e.g., a 3′UTR from an MCH or MCH-Rgene or from another gene; a polyadenylation site; an insulatorsequence. In another preferred embodiment, the level of an MCH or MCH-Rprotein is increased by increasing the level of expression of anendogenous MCH or MCH-R gene, e.g., by increasing transcription of theMCH or MCH-R gene or increasing MCH or MCH-R mRNA stability. In apreferred embodiment, transcription of the MCH or MCH-R gene isincreased by: altering the regulatory sequence of the endogenous MCH orMCH-R gene, e.g., by the addition of a positive regulatory element (suchas an enhancer or a DNA-binding site for a transcriptional activator);the deletion of a negative regulatory element (such as a DNA-bindingsite for a transcriptional repressor) and/or replacement of theendogenous regulatory sequence, or elements therein, with that ofanother gene, thereby allowing the coding region of the MCH or MCH-Rgene to be transcribed more efficiently.

In another embodiment, the agent decreases or inhibits MCH signaling. Inone embodiment, the agent decreases or inhibits MCH signaling by bindingto a protein on the surface of the β cell, e.g., MCH receptor, e.g.,MCH-R1 or SLC-1, and, e.g., inhibits MCH binding. An agent thatdecreases or inhibits MCH signaling can be one more of: an MCH or MCH-Rantagonist (e.g., a [D-Arg¹¹]-MCH); a soluble MCH binding protein, e.g.,a soluble MCH binding protein that binds to MCH and inhibits MCH bindingto MCH-R; a soluble MCH-R binding protein, e.g., a soluble MCH-R bindingprotein that binds to MCH-R and inhibits MCH-R binding to MCH or MCH-Rbinding to a secondary messenger, e.g., a G protein, e.g., G_(i) orG_(o); an antibody or antigen binding fragment thereof that specificallybinds to MCH or MCH-R, e.g., an antibody that disrupts MCH binding toMCH-R; a mutated inactive MCH-R (e.g., an Asp-123-substituted MCH-Rmutant) that, e.g., does not bind to MCH, or binds to MCH but disruptsan intracellular receptor signaling activity; an MCH or MCH-R nucleicacid molecule that can bind to a cellular MCH or MCH-R nucleic acidsequence, e.g., mRNA, and inhibit expression of the protein, e.g., anantisense molecule or MCH or MCH-R ribozyme; an agent which decreasesMCH or MCH-R gene expression, e.g., a small molecule which binds thepromoter of MCH or MCH-R and decreases MCH or MCH-R gene expression. Inanother preferred embodiment, MCH or MCH-R is inhibited by decreasingthe level of expression of an endogenous MCH or MCH-R gene, e.g., bydecreasing transcription of the MCH or MCH-R gene. In a preferredembodiment, transcription of the MCH or MCH-R gene can be decreased by:altering the regulatory sequences of the endogenous MCH or MCH-R gene,e.g., by the addition of a negative regulatory sequence (such as aDNA-biding site for a transcriptional repressor), or by the removal of apositive regulatory sequence (such as an enhancer or a DNA-binding sitefor a transcriptional activator).

In a preferred embodiment, the administration of the agent can beinitiated, e.g., (a) before a subject, e.g., a subject who is at riskfor an insulin relate disorder, shows clinical symptoms of an insulinrelated disorder; (b) after the subject begins to show signs of aninsulin related disorder, e.g., elevated glucose levels or β cellfailure (as evidenced, e.g., by an increase or decrease of more than 5,10, 20, or 30% in glucose levels or β cell failure compared to areference value, e.g., a control, e.g., a non-disease state control);(c) when an insulin related disease, e.g., diabetes or another insulinrelated disorder described herein is diagnosed; (d) before, during orafter a treatment for an insulin related disorder, e.g., diabetes, isbegun or begins to exert its effects. The period over which the agent isadministered (or the period over which clinically effective levels aremaintained in the subject) can be long term, e.g., for six months ormore or a year or more, or short term, e.g., for less than a year, sixmonths, one month, two weeks or less.

In a preferred embodiment, the agent is administered before the subjectshows clinical symptoms of an insulin related disorder, but after adetermination that the subject is at risk for an insulin relateddisorder, e.g., the subject is obese, or the subject has a familyhistory of insulin related disorder (e.g., a parent, sibling orgrandparent of the subject has an insulin related disorder).

In a preferred embodiment, the agent is administered in the early stagesof onset of clinical symptoms of an insulin related disorder, e.g.,diabetes, e.g., type 2 diabetes. For example, the agent is administeredwhen the subject begins to show elevated glucose levels or increased βcell dysfunction, but before complete β cell failure.

In a preferred embodiment, the agent is administered as a supplementaltherapy for an insulin related disorder, e.g., the agent is administeredin addition to administration of insulin.

In a preferred embodiment, the subject exhibits abnormal pancreaticfunction, e.g., abnormal insulin secretion, e.g., the subject has aninsulin related disorder, e.g., diabetes, e.g., type 1 or type 2diabetes; obesity; insulin resistance; hyperinsulinemia; hypoglycemia.

In a preferred embodiment, a pharmaceutical composition including one ormore of the agents described herein is administered in apharmaceutically effective dose.

In a preferred embodiment, a pharmaceutical composition including one ormore of the agents described herein is administered in a therapeuticallyeffective dose.

In a preferred embodiment, the subject is a non-human animal, e.g., ananimal model of an insulin related disorder, e.g., the NOD Mouse and itsrelated strains, BB Rat, Leptin or Leptin Receptor mutant rodents,Zucker Diabetic Fatty (ZDF) Rat, Sprague-Dawley rats, ObeseSpontaneously Hypertensive Rat (SHROB, Koletsky Rat), Wistar Fatty Rat,New Zealand Obese Mouse, NSY Mouse, Goto-Kakizaki Rat, OLETF Rat,JCR:LA-cp Rat, Neonatally Streptozotocin-Induced (n-STZ) Diabetic Rats,Rhesus Monkey, Psammomys obesus (fat sand rat), C57B1/6J Mouse.

In a preferred embodiment, the subject is a mammal, e.g., a human.

In a preferred embodiment, the subject is at risk for or has an insulinrelated disorder, e.g., an insulin related disorder described herein.

In a preferred embodiment, the method also includes evaluating thesubject for one or more of the following parameters: (1) insulin levels;(2) glucose levels; (3) weight; (4) endogenous MCH levels or activity;(5) endogenous MCH receptor (MCH-R) levels or activity.

In another aspect, the invention features a method of culturing orpropagating an islet cell or β cell preparation. The method includesculturing an islet cell or β cell preparation in the presence of MCH oran agent that increases or promotes MCH signaling, e.g., an agent thatincreases or promotes MCH signaling described herein, e.g., an MCHagonist. While not bound by theory, it is believed that MCH can act as agrowth factor for islet/β cells. In some embodiments, the islet cell orβ cell preparation includes a nucleic acid encoding an MCH-R, e.g.,MCH-R1, MCH-R2, SLT, or a functional fragment thereof.

In another aspect, the invention features a method of evaluating asubject, e.g., determining if a subject is at risk for, or has, aninsulin related disorder, e.g., an insulin related disorder describedherein. The method includes evaluating MCH signaling in a cell ortissue, preferably in the pancreas, islets, or β-cells, of the subject.Abnormal or aberrant MCH signaling as compared to a control can indicatethe risk or presence of an insulin related disorder, e.g., an insulinrelated disorder described herein. The method can include providing arecord, e.g., a print or computer readable material, e.g., aninformational, diagnostic, or instructional material, e.g., to thesubject, health care provider, or insurance company, identifying theabnormal or aberrant MCH signaling as a risk or diagnostic factor for aninsulin related disorder, e.g., an insulin related disorder describedherein.

In a preferred embodiment, the method includes detecting a geneticlesion or mutation in a gene involved in MCH signaling, in an MCH orMCH-R gene. The human MCH peptide gene sequence is available at, e.g.,Genbank Accession No. AI224977. The sequence of at least several humanMCH receptors (e.g., MCH-R1 and MCH-R2) is known, and is described,e.g., in Genbank Accession Nos. AF347063 and AF347063.

In a preferred embodiment, the method includes evaluating the level ofexpression of a gene involved in MCH signaling, e.g., in an MCH or MCH-Rgene, e.g., evaluating the amount or half life of an MCH or MCH-R mRNA.Over- or under-expression of a gene, compared to a control, can beevaluated by, e.g., Northern blot, TaqMan assay, or other methods knownin the art.

In a preferred embodiment, the method includes evaluating an MCHsignaling activity, e.g., MCH to MCH-R binding; or MCH-mediated insulinsecretion from β cells.

In a preferred embodiment, the method includes evaluating protein levelsof a protein involved in MCH signaling, e.g., levels of MCH-R, e.g.,SLC-1, in a β cell.

In a preferred embodiment, the method includes treating the subject forthe disorder.

In a preferred embodiment, the subject is further evaluated for one ormore of the following parameters: (1) insulin levels; (2) glucoselevels; (3) weight; (4) endogenous MCH levels or activity; (5)endogenous MCH receptor (MCH-R) levels or activity.

In a preferred embodiment, the evaluation is used to choose a course oftreatment.

Methods of the invention can be used prenatally or to determine if asubject's offspring will be at risk for a disorder.

In another aspect, the invention features a method of evaluating anagent, e.g., screening for an agent that modulates pancreatic function,e.g., β cell function, e.g., insulin secretion. The method includes (a)providing a test agent, (b) determining if the agent modulates MCHsignaling, e.g., interacts with a molecule involved in MCH signaling,e.g., MCH or MCH-R, e.g., binds to and/or modulates the levels,expression, or activity of MCH or MCH-R; and (c) correlating the abilityof a test agent to modulate MCH signaling with the ability to modulatepancreatic function (e.g., insulin production or secretion). Correlatingmeans identifying a test agent that modulates MCH signaling an agentcapable of modulating pancreatic function, e.g., providing a record,e.g., a print or computer readable record, such as a laboratory recordor dataset, identifying a test agent that modulates MCH signaling as anagent capable of modulating pancreatic function, e.g., insulinproduction or secretion. The record can include other information, suchas a specific test agent identifier, a date, an operator of the method,or information about the source, structure, method of purification orbiological activity of the test agent. The record or information derivedfrom the record can be used, e.g., to identify the test agent as acompound or lead compound for pharmaceutical or therapeutic use. Agents,e.g., compounds, identified by this method can be used, e.g., in thetreatment of an insulin related disorder, e.g., an insulin relateddisorder described herein.

In one embodiment, the method includes: providing an MCH or MCH-Rprotein or nucleic acid or a functional fragment thereof; contacting theMCH or MCH-R protein or nucleic acid with a test agent, and determiningif the test compound interacts with, e.g., binds, the MCH or MCH-Rprotein or nucleic acid.

In one embodiment, the test agent binds to the MCH or MCH-R protein andmodulates a MCH signaling activity. For example, the compound binds tothe MCH or MCH-R protein and facilitates or inhibits any of: MCH bindingto its receptor; intracellular MCH-R signaling, e.g., MCH-R binding to asecond messenger; insulin secretion. Methods for assaying MCH signaling,e.g., MCH activity, e.g., methods described herein, are art-recognized.

In a preferred embodiment, the test compound is one or more of: aprotein or peptide; an antibody or antigen-binding fragment thereof; asmall molecule; a nucleotide sequence. For example, the agent can be anagent identified through a library screen described herein.

In a preferred embodiment, the contacting step is performed in vitro.

In another preferred embodiment, the contacting step is performed invivo.

In a preferred embodiment, the method further includes administering thetest compound to an experimental animal, e.g., an animal model for aninsulin related disorder, e.g., an animal model disclosed herein.

In another embodiment, the method includes: providing a test cell,tissue, or subject; administering a test agent to the cell, tissue, orsubject; and determining whether the test agent modulates MCH signalingin the cell, tissue, or subject. An agent that is found to modulate,e.g., MCH or MCH-R in the cell, tissue, or subject is identified as anagent that can modulate pancreatic function, e.g., islet or β cellfunction, e.g., insulin secretion.

In a preferred embodiment, the cell is a β cell.

In a preferred embodiment, the MCH-R is a human MCH-R, e.g., MCH-R1 orMCH-R2.

In a preferred embodiment, the tissue is pancreatic tissue, e.g., anislet or islet equivalent.

In a preferred embodiment, the method includes (a) providing a cell-freeexpression system, cell, tissue, or animal having a transgene whichincludes a nucleic acid that encodes a reporter molecule functionallylinked to the control region, e.g., a promoter, of a gene encoding a MCHor MCH-R, e.g., an MCH-R described herein; (b) contacting the cell-freeexpression system, cell, tissue, or animal with a test agent; and (c)evaluating a signal produced by the reporter molecule. A test agent thatcauses the modulation of reporter molecule expression, compared to areference, e.g., a negative control, is identified as an agent that canmodulate pancreatic function, e.g., insulin function, e.g., insulinsecretion.

In a preferred embodiment, the reporter molecule is any of: greenfluorescent protein (GFP); enhanced GFP (EGFP); luciferase;chloramphenicol acetyl transferase (CAT); β-galactosidase; β-lactamase;or secreted placental alkaline phosphatase. Other reporter molecules,e.g., other enzymes whose function can be detected by appropriatechromogenic or fluorogenic substrates are known to those skilled in theart.

In a preferred embodiment, the agent is further tested in a cell-basedand/or animal based model e.g., a cell based or animal model describedherein.

In another aspect, the invention features a computer readable recordencoded with (a) a subject identifier, e.g., a patient identifier, (b)one or more results from an evaluation of the subject, e.g., adiagnostic evaluation described herein, e.g., the level of expression,level or activity of MCH or MCH-R, in the subject, and optionally (c) avalue for or related to a disease state, e.g., a value correlated withdisease status or risk with regard to an insulin related disorder, e.g.,an insulin related disorder described herein. In one embodiment, theinvention features a computer medium having a plurality of digitallyencoded data records. Each data record includes a value representing thelevel of expression, level or activity of MCH signaling, e.g., MCH orMCH-R levels or activity, in a sample, and a descriptor of the sample.The descriptor of the sample can be an identifier of the sample, asubject from which the sample was derived (e.g., a patient), adiagnosis, or a treatment (e.g., a preferred treatment). In a preferredembodiment, the data record further includes values representing thelevel of expression, level or activity of genes other than MCH or MCH-R(e.g., other genes associated with an insulin disorder, or other geneson an array). The data record can be structured as a table, e.g., atable that is part of a database such as a relational database (e.g., aSQL database of the Oracle or Sybase database environments). Theinvention also includes a method of communicating information about asubject, e.g., by transmitting information, e.g., transmitting acomputer readable record described herein, e.g., over a computernetwork.

In another aspect, the invention features a method of providinginformation, e.g., for making a decision with regard to the treatment ofa subject having, or at risk for, an insulin disorder described herein.The method includes (a) evaluating the expression, level or activity ofMCH or MCH-R; optionally (b) providing a value for the expression, levelor activity of MCH or MCH-R; optionally (c) comparing the provided valuewith a reference value, e.g., a control or non-disease state referenceor a disease state reference; and optionally (d) based, e.g., on therelationship of the provided value to the reference value, supplyinginformation, e.g., information for making a decision on or related tothe treatment of the subject.

In a preferred embodiment, the provided value relates to an activitydescribed herein, e.g., to a binding activity of MCH or MCH-R.

In a preferred embodiment, the decision is whether to administer apreselected treatment.

In a preferred embodiment, the decision is whether a party, e.g., aninsurance company, HMO, or other entity, will pay for all or part of apreselected treatment.

Also featured is a method of evaluating a sample. The method includesproviding a sample, e.g., from the subject, and determining a geneexpression profile of the sample, wherein the profile includes a valuerepresenting the level of expression of a MCH or MCH-R. The method canfurther include comparing the value or the profile (i.e., multiplevalues) to a reference value or reference profile. The gene expressionprofile of the sample can be obtained by methods known in the art (e.g.,by providing a nucleic acid from the sample and contacting the nucleicacid to an array). The method can be used to diagnose an insulin relateddisorder, e.g., an insulin related disorder described herein, in asubject wherein misexpression of an MCH signaling molecule, e.g., MCH orMCH-R, is an indication that the subject has or is disposed to having aninsulin related disorder, e.g., an insulin related disorder describedherein. The method can be used to monitor a treatment for an insulinrelated disorder in a subject. For example, the gene expression profilecan be determined for a sample from a subject undergoing treatment. Theprofile can be compared to a reference profile or to a profile obtainedfrom the subject prior to treatment or prior to onset of the disorder(see, e.g., Golub et al. (1999) Science 286:531).

In another aspect, the invention features a method of evaluating a genefor its involvement in an insulin related disorder, e.g., in an insulinrelated disorder described herein. The method includes (a) providing acell, tissue, or animal in which MCH signaling is perturbed, e.g., MCHor MCH-R is perturbed, (b) evaluating the expression of one or moregenes in the cell, tissue, or animal, and (c) optionally comparing theexpression of the one or more genes in the cell, tissue, or animal witha reference, e.g., with the expression of the one or more genes in acontrol cell, tissue or animal. A gene or genes identified as increasedor decreased in the cell, tissue, or animal as compared to thereference, e.g., the control, are identified as candidate genes involvedin an insulin related disorder, e.g., an insulin related disorderdescribed herein.

In a preferred embodiment, the cell or tissue is from a subject (e.g., ahuman or non-human animal, e.g., an experimental animal) having or beingat risk for an insulin disorder, e.g., an insulin disorder describedherein.

In a preferred embodiment, the animal is a transgenic animal, e.g., atransgenic animal having a knock-out or overexpressing mutation for acomponent of the MCH signaling pathway, e.g., MCH or MCH-R.

In yet another aspect, the invention features a method of evaluating atest compound, e.g., evaluating a test compound for the ability tomodulate MCH signaling. The method includes providing or obtaining acell or tissue that naturally expresses MCH and MCHR, e.g., an isletcell or tissue, e.g., an islet cell or tissue (e.g., a RINm5F or bTC3cell line); providing or obtaining a test compound; and evaluating theability of the test compound to modulate MCH signaling in the islet cellor tissue. The method can be performed using routine screeningtechniques. In preferred embodiments, the islet cell or tissue is nottransfected with an MCH or MCHR transgene.

In yet another aspect, the invention features a method of evaluating atest compound. The method includes providing a cell and a test compound;contacting the test compound to the cell; obtaining a subject expressionprofile for the contacted cell; and comparing the subject expressionprofile to one or more reference profiles. The profiles include a valuerepresenting the level of expression of a component of the MCH signalingpathway, e.g., MCH or MCH-R. In a preferred embodiment, the subjectexpression profile is compared to a target profile, e.g., a profile fora normal cell or for desired condition of a cell. The test compound isevaluated favorably if the subject expression profile is more similar tothe target profile than an expression profile obtained from anuncontacted cell.

In another aspect, the invention features, a method of evaluating asubject. The method includes: a) obtaining a sample from a subject,e.g., from a caregiver, e.g., a caregiver who obtains the sample fromthe subject; b) determining a subject expression profile for the sample.Optionally, the method further includes either or both of steps: c)comparing the subject expression profile to one or more referenceexpression profiles; and d) selecting the reference profile most similarto the subject reference profile. The subject expression profile and thereference profiles include a value representing the level of expressionof a component of the MCH signaling pathway, e.g., MCH or MCH-R. Avariety of routine statistical measures can be used to compare tworeference profiles. One possible metric is the length of the distancevector that is the difference between the two profiles. Each of thesubject and reference profile is represented as a multi-dimensionalvector, wherein each dimension is a value in the profile.

The method can further include transmitting a result to a caregiver. Theresult can be the subject expression profile, a result of a comparisonof the subject expression profile with another profile, a most similarreference profile, or a descriptor of any of the aforementioned. Theresult can be transmitted across a computer network, e.g., the resultcan be in the form of a computer transmission, e.g., a computer datasignal embedded in a carrier wave.

Also featured is a computer medium having executable code for effectingthe following steps: receive a subject expression profile; access adatabase of reference expression profiles; and either i) select amatching reference profile most similar to the subject expressionprofile or ii) determine at least one comparison score for thesimilarity of the subject expression profile to at least one referenceprofile. The subject expression profile, and the reference expressionprofiles each include a value representing the level of expression of acomponent of the MCH signaling pathway, e.g., MCH or MCH-R.

As used herein, “treatment” or “treating a subject” is defined as theapplication or administration of a therapeutic agent to a patient, orapplication or administration of a therapeutic agent to an isolatedtissue or cell line from a patient, e.g., a pancreatic tissue, e.g., anislet tissue, or β-cell, who has a disease, a symptom of disease or apredisposition toward a disease, e.g., an insulin related disorder,e.g., an insulin disorder described herein. Treatment can slow, cure,heal, alleviate, relieve, alter, remedy, ameliorate, improve or affectthe disease, a symptom of the disease or the predisposition towarddisease, e.g., by at least 10%.

As used herein, to ability of a first molecule to “interact” with asecond molecule refers to the ability of the first molecule to act uponthe structure and/or activity of the second molecule, either directly orindirectly. For example, a first molecule can interact with a second by(a) directly binding, e.g., specifically binding, the second molecule,e.g., transiently or stably binding the second molecule; (b) modifyingthe second molecule, e.g., by cleaving a bond, e.g., a covalent bond, inthe second molecule, or adding or removing a chemical group to or fromthe second molecule, e.g., adding or removing a phosphate group orcarbohydrate group; (c) modulating an enzyme that modifies the secondmolecule, e.g., inhibiting or activating a kinase or phosphatase thatnormally modifies the second molecule; (d) affecting expression of thesecond molecule, e.g., by binding, activating, or inhibiting a controlregion of a gene encoding the second molecule, or binding, activating,or inhibiting a transcription factor that associates with the geneencoding the second molecule; (d) affecting the stability of an mRNAencoding the second molecule, e.g., by inhibiting mRNAse activityagainst the mRNA encoding the second molecule or by degrading the mRNAencoding the second molecule.

DETAILED DESCRIPTION

The inventors have found that overexpression of MCH results in increasedplasma insulin concentration and increased pancreatic islet size. Thedata described herein indicate that MCH stimulates insulin release fromthe islets/β-cells via its own receptor. The inventors have thus foundthat components of the MCH signaling pathway, e.g., MCH or MCH-R (e.g.,MCH-R1 or MCH-R2) are targets for the diagnosis and treatment of insulinrelated disorders, e.g., insulin related disorders described herein.

Analogs Of MCH

The sequence of the cyclic MCH peptide is as follows.NH2-Asp-Phe-Asp-Met-Leu-Arg-Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Gln-Val-COOH(SEQ ID NO:1). The importance of the disulfide bond for activity hasbeen demonstrated by chemical reduction and by the synthesis of ringcontraction analogs, both of which eliminated ligand activity. Chemicalmodification of either the Tyr or Arg residues significantly reducesactivity. Truncation at either the carboxy or amino terminus results inno loss of activity, with the minimum peptide analog retaining thepotency of the native peptide being MCH(5-15). Arg11 has been identifiedas a requisite residue for binding of human MCH to its receptor,confirming the importance of the cyclic core of MCH as the majorpharmacophore of MCH receptor function (Macdonald et al. (2000) MolPharmacol 58(1):217-25).

Synthetic MCH and its analogs can be prepared using art-recognizedmethods to identify agonists and antagonists, and determine thestructural requirements for MCH agonist or antagonist activity. MCH canbe modified in a number of ways, e.g., by shortening either (or both)the amino- or carboxy-terminal regions, contracting the cysteine bridgedring, forming acyclic analogs, or modifying or substituting an aminoacid, e.g., a residue, within, or outside, the ring. Synthetic MCH andits analogs can be assayed using one or more of the assays describedherein.

Generally, the synthetic schemes use the Merrifield solid phasesynthesis followed by cyclization and purification as described, e.g.,in Lebl et al. (1988) J. Med. Chem. 31:949-954, herein incorporated byreference. Briefly, chloromethylated resin can be used as the support tointroduce the first amino acid on an automated synthesizer, e.g. aDuPont 2200. The intact peptides are cleaved from the resin and thenwashed. Following extraction from the wash the peptides are lyophilized.The lyophilized protein is dissolved in degassed water. Cyclization isachieved by the dropwise addition of potassium ferricyanide (K.sub.3Fe(CN).sub.6). Purification can be performed by column chromatography onSephadex G-25, carboxymethyl cellulose and by reversed-phasehigh-performance chromatography (HPLC).

Alternatively, truncated MCH analogs can be prepared by exposing naturalor synthetic MCH to enzymes. Natural MCH can be isolated frompituitaries using an acetone extraction and purified on an HPLC columnas described in Kawauchi et al. (1988) Adv. in Pigment Cell Res.517-530, herein incorporated by reference. For example, MCH.sub.1-14, acarboxy-terminal truncation, can be generated from MCH by exposure tocarboxypeptidase Y.

Acyclic analogs can be constructed by replacing the Cys.sup.5-Cys.sup.14bridge with pseudoisosteric residues. Either L-serine, a polarsubstitute, or L-.alpha.-aminobutyrate, a non-polar substitute, can beutilized. The peptides, with the appropriate substitution, can beprepared by solid phase synthesis as described above and in Matsunaga etal., Life Sci. (1992) 51:679-685, herein incorporated by reference.

Modification of amino acids within the ring is performed with a reagentspecific to each residue. Modifications can be accomplished either bysubstituting a different amino acid or altering the existing amino acid.For example, Arg (11) is a critical residue for MCH function (Macdonaldet al. (2000) Mol Pharmacol 58(1):217-25). The Tyr residue at position11 can be modified with the addition of a —NO₂ group by exposing MCH toa solution of 10% nitromethane-95% ethanol. See, e.g., Kawauchi et al.(1988) Adv. in Pigment Cell Res. 517-530, herein incorporated byreference.

Structural Requirements for MCH Activity

A significant amount of work has been done on determining structuralrequirements for MCH. The ring structure of MCH has been found in anumber of studies to be important for activity.

Numerous investigators have synthesized N-terminal and C-terminalfragment analogs of salmon MCH and have tested them for MCH activity inteleost skin bioassay and frog and lizard bioassays, described herein(see, e.g., Matsunaga et al. (1989) Peptides 10:349-354; Hardley et al.(1987) Life Sci. 40:1139-1145, herein incorporated by reference). Thesestudies have concluded that the minimal sequence needed to elicit anequipotent response to the native MCH is MCH(5-15), a structure whichlacks residues 1-4 from the N-terminal end, and residues 16-17 of theC-terminal end of the peptide. The removal of Trp¹⁵, producing afragment MCH(5-14), results in an analog 100 to 300 less active thannative MCH indicating that Trp at position 15 is important formaintenance of full (equipotent) agonist activity of MCH, and thatindole ring of Trp residue may be important in aiding the fit of MCHinto its receptor pocket, thus facilitating binding. Because fragmentanalogs, which are N terminal deleted, e.g., those lacking residues 1-4,are equipotent to native MCH, they appear to not be required for MCHactivity. The same was concluded for residues 16-17 in the C-terminalend of the peptide.

Furthermore, other investigators have synthesized MCH analogs withcontracted ring structure and have tested them for activity in teleostfish skin bioassay (see, e.g., Lebl et al. (1988) J. Med. Chem.31:949-954; Lebl et al. (1989) Life Sci. 44:451-457; Matsunaga et al.(1989) Peptides 10:349-354, herein incorporated by reference). Thefollowing ring contraction analogs (which retain a disulfide bond) weresynthesized: [Ala⁵, Cys¹⁰]MCH, [Ala⁵, Cys⁸]MCH, [Ala⁵, Cys⁷]MCH, [Ala⁵,Cys¹⁰]MCH₅₋₁₇, [Ala⁵, Cys⁸]MCH₅₋₁₇, [Ala⁵, Cys⁷]MCH₅₋₁₇,[Cys¹⁰]MCH₁₀₋₁₇, [Cys⁸]MCH₈₋₁₇, and [Cys⁷]MCH₇₋₁₇. The studies withthese analogs have concluded that the disulfide bond between positions 5and 14 is essential for the MCH-like activity, because ring contractionseliminated or greatly reduced the MCH-like activity. It seems that the10 ring residue structure, MCH(5-14) is very important for optimalactivation. Surprisingly, two of the analogs, [Ala⁵, Cys⁸]MCH₅₋₁₇ and[Cys¹⁰]MCH₁₀₋₁₇, were found to be full agonists, however, with veryreduced potency, indicating that the shortest sequence having MCH-likeactivity may be comprised of residues 10-14 (Val-Tyr-Arg-Pro-Cys;residues 12-16 of SEQ ID NO:1) with residues at positions 11-14(Tyr-Arg-Pro-Cys; residues 13-16 of SEQ ID NO:1) possibly being crucialfor message transduction.

In addition, acyclic analogs have been synthesized and tested for MCHactivity in teleost fish skin bioassay (see, e.g., Kawauchi and Kawazoe(1988) Advances in Pigment Cell Res. 517-527; Matsunaga et al. (1992)Life Sci. 51:679-685, herein incorporated by reference). These analogswere constructed so that they differed form native MCH only in thepolarity of the side chain group at positions 5 and 14. For one analogpolar L-serine was substituted for cysteine at positions 5 and 14(L-Ser^(5,14) MCH), while for the other analog, non-polar Lα-aminobutyrate (Abu) was substituted at the same positions (Abu^(5,14)MCH). Another acyclic analog was constructed by reduction of thedisulfide bond, followed by subsequent carboxymethylation of Cysresidues at positions 5 and 14 (CAM-Cys^(5,14) MCH). All of theseanalogs exhibited no MCH-like activity, suggesting that the disulfidebridge is necessary to maintain correct conformation and topographicalfeatures of MCH for receptor binding and transmembrane signaltransduction.

MCH derivatives with modified residues have also been synthesized andtested for activity in fish scale assay (see, e.g., Kawauchi and Kawazoe(1988) Advances in Pigment Cell Res. 517-527, herein incorporated byreference). The following derivatives have been synthesized and testedfor activity: NPS-Trp¹⁵MCH, DHCH-Arg^(4,9,12)MCH, NO₂-Tyr¹¹MCH andS-O-Met^(3,6)MCH. Modifications of amino acid residues outside of thering structure had no effect on the MCH activity, while themodifications of residues within the ring, e.g., DHCH-Arg^(4,9,12)MCH,NO₂-Tyr¹¹MCH and S-O-Met^(3,6)MCH, resulted in analogs with greatlyreduced MCH activity. These results support the suggestion that the MCHactivity is elicited from the cyclic segment (MCH5-14) of the peptide.Indeed, a compound consisting merely of the cyclic core of human MCHwith the Arg attached to the N-terminus of the disulfide ring(Arg-cyclo(S-S)(Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys) (SEQ ID NO:2)can activate both human MCH-1R and human MCH-2R receptors about aseffectively as full-length human MCH (Bednarek et al. (2001)Biochemistry 40(31):9379-86). Selective antagonists, e.g., for hMCH-1R,are also known in the art (see, e.g., Bednarek et al. (2002)Biochemistry 41(20):6383-90; and Borowsky et al. (2002) Nat Med8(8):825-30).

Audinot et al. (J Biol Chem. (2001) 276(17):13554-62) made numerousalanine scanning peptide analogs of MCH and tested the mutant peptidesfor activity against a human cell transfected with a human MCH receptor.Using this assay system, Audinot et al. found numerous antagonists (8 of57 mutant peptides made). All of the antagonists included changes in theMCH ring structure.

Non-peptide antagonists or agonists of MCH are also known and can bereadily identified. For example, Takekawa et al. (2002) European J.Pharmacol. 438:129-135 used a combination of in vitro and in vivotesting to identify the MCH antagonist T-226296, a (−) enantiomer ofN-[6-(dimethylamino)-methyl]-5,6,7,8-tetrahydro-2-naphthalenyl]-4′-fluoro[1,1′-biphenyl]-4-carboxamide,from a library of chemical compounds.

Analogs of MCH Receptor

Human MCH receptors and analogs and variants thereof are described in:Chambers et al. (1999) Nature 400(6741):261-5 and Saito et al. (1999)Nature 400(6741):265-9 (MCH-R1); Rodriguez et al. (2001) Mol Pharmacol.60(4):632-9, Wang et al. (2001) J Biol Chem. 276(37):34664-70, Sailer etal. (2001) Proc Natl Acad Sci U S A 98(13):7564-9 and Hill et al (2001)J. Biol. Chem. 276 (23),:20125-20129 (MCH-R2); Mori et a (2001) BiochemBiophys Res Commun. 283(5):1013-8 (SLT); and U.S. Pat. Nos. 6,291,195;6,221,616; and 6,221,613, all of which are incorporated herein byreference. The amino acid and nucleotide (coding) sequence of the humanMCH-R1 (also known as SLC-1) and MCH-R2 receptors can be found online(GenBank Accession No. AB063174 and AF347063, respectively). For areview of MCH receptors, see Boutin et al. (2002) Can J PhysiolPharmacol 80(5):388-95.

Critical residues involved in binding and activation of the MCH/receptorcomplex are identified in Macdonald et al. (2000) Mol Pharmacol58(1):217-25. E.g., Macdonald et al. conclude that Asp(123)(3.32) in theMCH receptor is required for the formation of the MCH peptide/receptorcomplex and form a direct interaction that is critical for receptorfunction.

Assays for MCH Signaling Activity

The activity of compounds, e.g., MCH analogs, e.g., MCH agonists orantagonists, and MCH receptor analogs, e.g., MCH receptor agonists orantagonists, can be determined by a number of in vitro, ex-vivo and invivo assays for MCH signaling activity known in the art. Examples ofsuch assays can be found, e.g., in U.S. Pat. No. 5,849,708; Audinot etal. (2001) J Biol Chem. 276(17):13554-62; and Macdonald et al. (2000)Mol Pharmacol 58(1):217-25, all of which are incorporated herein byreference. Furthermore, the activity of an analog, e.g., an MCH or MCHreceptor agonist or antagonist described herein, can be determined inthe methods described herein by evaluating the ability of the subjectanalog to stimulate insulin release from pancreas, isolated islets orislet equivalents, isolated β-cells, or in vivo in an animal, e.g., arodent. Insulin secretion, e.g., from an islet or β-cell, can bemeasured by, e.g., standard detection techniques, including enzymelinked immunosorbent assays (ELISAs), immunoprecipitations,immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA),and Western blot analysis.

Generation of Variants: Production of Altered DNA and Peptide Sequencesby Random Methods

Amino acid sequence variants of components of the MCH signaling pathway,e.g., MCH or MCH-R, or fragments thereof, can be prepared by randommutagenesis of DNA which encodes a component of the MCH signalingpathway, e.g., MCH or MCH-R or a region thereof. Useful methods includePCR mutagenesis and saturation mutagenesis, as described below. Alibrary of random amino acid sequence variants can also be generated bythe synthesis of a set of degenerate oligonucleotide sequences.

PCR Mutagenesis

In PCR mutagenesis, reduced Taq polymerase fidelity is used to introducerandom mutations into a cloned fragment of DNA (Leung et al., 1989,Technique 1:11-15). This is a very powerful and relatively rapid methodof introducing random mutations. The DNA region to be mutagenized isamplified using the polymerase chain reaction (PCR) under conditionsthat reduce the fidelity of DNA synthesis by Taq DNA polymerase, e.g.,by using a dGTP/dATP ratio of five and adding Mn²⁺ to the PCR reaction.The pool of amplified DNA fragments are inserted into appropriatecloning vectors to provide random mutant libraries.

Saturation Mutagenesis

Saturation mutagenesis allows for the rapid introduction of a largenumber of single base substitutions into cloned DNA fragments (Mayers etal., 1985, Science 229:242). This technique includes generation ofmutations, e.g., by chemical treatment or irradiation of single-strandedDNA in vitro, and synthesis of a complimentary DNA strand. The mutationfrequency can be modulated by modulating the severity of the treatment,and essentially all possible base substitutions can be obtained. Becausethis procedure does not involve a genetic selection for mutant fragmentsboth neutral substitutions, as well as those that alter function, areobtained. The distribution of point mutations is not biased towardconserved sequence elements.

Degenerate Olizonucleotides

A library of homologs can also be generated from a set of degenerateoligonucleotide sequences. Chemical synthesis of a degenerate sequencescan be carried out in an automatic DNA synthesizer, and the syntheticgenes then ligated into an appropriate expression vector. The synthesisof degenerate oligonucleotides is known in the art (see for example,Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) RecombinantDNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. A G Walton,Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem.53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)Nucleic Acid Res. 11:477. Such techniques have been employed in thedirected evolution of other proteins (see, for example, Scott et al.(1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433;Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

Generation of Variants: Production of Altered DNA and Peptide Sequencesby Directed Mutagenesis

Non-random or directed, mutagenesis techniques can be used to providespecific sequences or mutations in specific regions. These techniquescan be used to create variants which include, e.g., deletions,insertions, or substitutions, of residues of the known amino acidsequence of a protein. The sites for. mutation can be modifiedindividually or in series, e.g., by (1) substituting first withconserved amino acids and then with more radical choices depending uponresults achieved, (2) deleting the target residue, or (3) insertingresidues of the same or a different class adjacent to the located site,or combinations of options 1-3.

Alanine Scanning Mutagenesis

Alanine scanning mutagenesis is a useful method for identification ofcertain residues or regions of the desired protein that are preferredlocations or domains for mutagenesis, Cunningham and Wells (Science244:1081-1085, 1989). In alanine scanning, a residue or group of targetresidues are identified (e.g., charged residues such as Arg, Asp, His,Lys, and Glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine). Replacement of an amino acidcan affect the interaction of the amino acids with the surroundingaqueous environment in or outside the cell. Those domains demonstratingfunctional sensitivity to the substitutions are then refined byintroducing further or other variants at or for the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to optimize the performance of amutation at a given site, alanine scanning or random mutagenesis may beconducted at the target codon or region and the expressed desiredprotein subunit variants are screened for the optimal combination ofdesired activity.

Oligonucleotide-Mediated Mutagenesis

Oligonucleotide-mediated mutagenesis is a useful method for preparingsubstitution, deletion, and insertion variants of DNA, see, e.g.,Adelman et al., (DNA 2:183, 1983). Briefly, the desired DNA is alteredby hybridizing an oligonucleotide encoding a mutation to a DNA template,where the template is the single-stranded form of a plasmid orbacteriophage containing the unaltered or native DNA sequence of thedesired protein. After hybridization, a DNA polymerase is used tosynthesize an entire second complementary strand of the template thatwill thus incorporate the oligonucleotide primer, and will code for theselected alteration in the desired protein DNA. Generally,oligonucleotides of at least 25 nucleotides in length are used. Anoptimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al. (Proc.NatL. Acad. Sci. (1978) USA, 75: 5765).

Cassette Mutagenesis

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al. (Gene, 1985, 34:315). Thestarting material is a plasmid (or other vector) which includes theprotein subunit DNA to be mutated. The codon(s) in the protein subunitDNA to be mutated are identified. There must be a unique restrictionendonuclease site on each side of the identified mutation site(s). If nosuch restriction sites exist, they may be generated using theabove-described oligonucleotide-mediated mutagenesis method to introducethem at appropriate locations in the desired protein subunit DNA. Afterthe restriction sites have been introduced into the plasmid, the plasmidis cut at these sites to linearize it. A double-stranded oligonucleotideencoding the sequence of the DNA between the restriction sites butcontaining the desired mutation(s) is synthesized using standardprocedures. The two strands are synthesized separately and thenhybridized together using standard techniques. This double-strandedoligonucleotide is referred to as the cassette. This cassette isdesigned to have 3′ and 5′ ends that are comparable with the ends of thelinearized plasmid, such that it can be directly ligated to the plasmid.This plasmid now contains the mutated desired protein subunit DNAsequence.

Combinatorial Mutagenesis

Combinatorial mutagenesis can also be used to generate mutants. Forexample, the amino acid sequences for a group of homologs or otherrelated proteins are aligned, preferably to promote the highest homologypossible. All of the amino acids which appear at a given position of thealigned sequences can be selected to create a degenerate set ofcombinatorial sequences. The variegated library of variants is generatedby combinatorial mutagenesis at the nucleic acid level, and is encodedby a variegated gene library. For example, a mixture of syntheticoligonucleotides can be enzymatically ligated into gene sequences suchthat the degenerate set of potential sequences are expressible asindividual peptides, or alternatively, as a set of larger fusionproteins containing the set of degenerate sequences.

Primary High-Through-Put Methods for Screening Libraries of PeptideFragments or Homologs

Various techniques are known in the art for screening peptides, e.g.,synthetic peptides, antibodies or antigen binding fragments thereof,small molecular weight peptides (e.g., linear or cyclic peptides) orgenerated mutant gene products. Techniques for screening large genelibraries often include cloning the gene library into replicableexpression vectors, transforming appropriate cells with the resultinglibrary of vectors, and expressing the genes under conditions in whichdetection of a desired activity, e.g., binding to a natural ligand,e.g., a receptor or substrate, facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Each of thetechniques described below is amenable to high through-put analysis forscreening large numbers of sequences created, e.g., by randommutagenesis techniques.

Two Hybrid Systems

Two hybrid (interaction trap) assays can be used to identify a proteinthat interacts with a component of the MCH signaling pathway, e.g., MCHor MCH-R or active fragments thereof. These may include, e.g., agonists,superagonists, and antagonists of MCH signaling. (The subject proteinand a protein it interacts with are used as the bait protein and fishproteins.). These assays rely on detecting the reconstitution of afunctional transcriptional activator mediated by protein-proteininteractions with a bait protein. In particular, these assays make useof chimeric genes which express hybrid proteins. The first hybridcomprises a DNA-binding domain fused to the bait protein, e.g., acomponent of the MCH signaling pathway, e.g., MCH or MCH-R or activefragments thereof. The second hybrid protein contains a transcriptionalactivation domain fused to a “fish” protein, e.g. an expression library.If the fish and bait proteins are able to interact, they bring intoclose proximity the DNA-binding and transcriptional activator domains.This proximity is sufficient to cause transcription of a reporter genewhich is operably linked to a transcriptional regulatory site which isrecognized by the DNA binding domain, and expression of the marker genecan be detected and used to score for the interaction of the baitprotein with another protein.

Display Libraries

In one approach to screening assays, the candidate peptides aredisplayed on the surface of a cell or viral particle, and the ability ofparticular cells or viral particles to bind an appropriate receptorprotein via the displayed product is detected in a “panning assay”. Forexample, the gene library can be cloned into the gene for a surfacemembrane protein of a bacterial cell, and the resulting fusion proteindetected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991)Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS 18:136-140).This technique was used in Sahu et al. (1996) J. Immunology 157:884-891,to isolate an inhibitor of a target protein. In a similar fashion, adetectably labeled ligand can be used to score for potentiallyfunctional peptide homologs. Fluorescently labeled ligands, e.g.,receptors, can be used to detect homolog which retain ligand-bindingactivity. The use of fluorescently labeled ligands, allows cells to bevisually inspected and separated under a fluorescence microscope, or,where the morphology of the cell permits, to be separated by afluorescence-activated cell sorter.

A gene library can be expressed as a fuision protein on the surface of aviral particle. For instance, in the filamentous phage system, foreignpeptide sequences can be expressed on the surface of infectious phage,thereby conferring two significant benefits. First, since these phagecan be applied to affinity matrices at concentrations well over 10¹³phage per milliliter, a large number of phage can be screened at onetime. Second, since each infectious phage displays a gene product on itssurface, if a particular phage is recovered from an affinity matrix inlow yield, the phage can be amplified by another round of infection. Thegroup of almost identical E. coli filamentous phages M13, fd., and flare most often used in phage display libraries. Either of the phage gIIIor gVIII coat proteins can be used to generate fusion proteins withoutdisrupting the ultimate packaging of the viral particle. Foreignepitopes can be expressed at the NH2-terminal end of pIII and phagebearing such epitopes recovered from a large excess of phage lackingthis epitope (Ladner et al. PCT publication WO 90/02909; Garrard et al.,PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem.267:16007-16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson etal. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS89:4457-4461).

A common approach uses the maltose receptor of E. coli (the outermembrane protein, LamB) as a peptide fuision partner (Charbit et al.(1986) EMBO 5, 3029-3037). Oligonucleotides have been inserted intoplasmids encoding the LamB gene to produce peptides fused into one ofthe extracellular loops of the protein. These peptides are available forbinding to ligands, e.g., to antibodies, and can elicit an immuneresponse when the cells are administered to animals. Other cell surfaceproteins, e.g., OmpA (Schorr et al. (1991) Vaccines 91, pp. 387-392),PhoE (Agterberg, et al. (1990) Gene 88, 37-45), and PAL (Fuchs et al.(1991) Bio/Tech 9, 1369-1372), as well as large bacterial surfacestructures have served as vehicles for peptide display. Peptides can befused to pilin, a protein which polymerizes to form the pilus-a conduitfor interbacterial exchange of genetic information (Thiry et al. (1989)Appl. Environ. Microbiol. 55, 984-993). Because of its role ininteracting with other cells, the pilus provides a useful support forthe presentation of peptides to the extracellular environment. Anotherlarge surface structure used for peptide display is the bacterial motiveorgan, the flagellum. Fusion of peptides to the subunit proteinflagellin offers a dense array of may peptides copies on the host cells(Kuwajima et al. (1988) Bio/Tech. 6, 1080-1083). Surface proteins ofother bacterial species have also served as peptide fusion partners.Examples include the Staphylococcus protein A and the outer membraneprotease IgA of Neisseria (Hansson et al. (1992) J. Bacteriol. 174,4239-4245 and Klauser et al. (1990) EMBO J. 9, 1991-1999).

In the filamentous phage systems and the LamB system described above,the physical link between the peptide and its encoding DNA occurs by thecontainment of the DNA within a particle (cell or phage) that carriesthe peptide on its surface. Capturing the peptide captures the particleand the DNA within. An alternative scheme uses the DNA-binding proteinLacI to form a link between peptide and DNA (Cull et al. (1992) PNAS USA89:1865-1869). This system uses a plasmid containing the LacI gene withan oligonucleotide cloning site at its 3′-end. Under the controlledinduction by arabinose, a LacI-peptide fusion protein is produced. Thisfusion retains the natural ability of LacI to bind to a short DNAsequence known as LacO operator (LacO). By installing two copies of LacOon the expression plasmid, the LacI-peptide fusion binds tightly to theplasmid that encoded it. Because the plasmids in each cell contain onlya single oligonucleotide sequence and each cell expresses only a singlepeptide sequence, the peptides become specifically and stably associatedwith the DNA sequence that directed its synthesis. The cells of thelibrary are gently lysed and the peptide-DNA complexes are exposed to amatrix of immobilized receptor to recover the complexes containingactive peptides. The associated plasmid DNA is then reintroduced intocells for amplification and DNA sequencing to determine the identity ofthe peptide ligands. As a demonstration of the practical utility of themethod, a large random library of dodecapeptides was made and selectedon a monoclonal antibody raised against the opioid peptide dynorphin B.A cohort of peptides was recovered, all related by a consensus sequencecorresponding to a six-residue portion of dynorphin B. (Cull et al.(1992) Proc. Natl. Acad. Sci. U.S.A. 89-1869)

This scheme, sometimes referred to as peptides-on-plasmids, differs intwo important ways from the phage display methods. First, the peptidesare attached to the C-terminus of the fusion protein, resulting in thedisplay of the library members as peptides having free carboxy termini.Both of the filamentous phage coat proteins, pIII and pVIII, areanchored to the phage through their C-termini, and the guest peptidesare placed into the outward-extending N-terminal domains. In somedesigns, the phage-displayed peptides are presented right at the aminoterminus of the fusion protein. (Cwirla, et al. (1990) Proc. Natl. Acad.Sci. U.S.A. 87, 6378-6382) A second difference is the set of biologicalbiases affecting the population of peptides actually present in thelibraries. The LacI fusion molecules are confined to the cytoplasm ofthe host cells. The phage coat fusions are exposed briefly to thecytoplasm during translation but are rapidly secreted through the innermembrane into the periplasmic compartment, remaining anchored in themembrane by their C-terminal hydrophobic domains, with the N-termini,containing the peptides, protruding into the periplasm while awaitingassembly into phage particles. The peptides in the LacI and phagelibraries may differ significantly as a result of their exposure todifferent proteolytic activities. The phage coat proteins requiretransport across the inner membrane and signal peptidase processing as aprelude to incorporation into phage. Certain peptides exert adeleterious effect on these processes and are underrepresented in thelibraries (Gallop et al. (1994) J. Med. Chem. 37(9):1233-1251). Theseparticular biases are not a factor in the LacI display system.

The number of small peptides available in recombinant random librariesis enormous. Libraries of 10⁷-10⁹ independent clones are routinelyprepared. Libraries as large as 10¹¹ recombinants have been created, butthis size approaches the practical limit for clone libraries. Thislimitation in library size occurs at the step of transforming the DNAcontaining randomized segments into the host bacterial cells. Tocircumvent this limitation, an in vitro system based on the display ofnascent peptides in polysome complexes has recently been developed. Thisdisplay library method has the potential of producing libraries 3-6orders of magnitude larger than the currently available phage/phagemidor plasmid libraries. Furthermore, the construction of the libraries,expression of the peptides, and screening, is done in an entirelycell-free format.

In one application of this method (Gallop et al. (1994) J. Med. Chem.37(9):1233-1251), a molecular DNA library encoding 10¹² decapeptides wasconstructed and the library expressed in an E. coli S30 in vitro coupledtranscription/translation system. Conditions were chosen to stall theribosomes on the mRNA, causing the accumulation of a substantialproportion of the RNA in polysomes and yielding complexes containingnascent peptides still linked to their encoding RNA. The polysomes aresufficiently robust to be affinity purified on immobilized receptors inmuch the same way as the more conventional recombinant peptide displaylibraries are screened. RNA from the bound complexes is recovered,converted to cDNA, and amplified by PCR to produce a template for thenext round of synthesis and screening. The polysome display method canbe coupled to the phage display system. Following several rounds ofscreening, cDNA from the enriched pool of polysomes was cloned into aphagemid vector. This vector serves as both a peptide expression vector,displaying peptides fused to the coat proteins, and as a DNA sequencingvector for peptide identification. By expressing the polysome-derivedpeptides on phage, one can either continue the affinity selectionprocedure in this format or assay the peptides on individual clones forbinding activity in a phage ELISA, or for binding specificity in acompletion phage ELISA (Barret, et al. (1992) Anal. Biochem204,357-364). To identify the sequences of the active peptides onesequences the DNA produced by the phagemid host.

Secondary Screens for Modulators of MCH Signaling

The high through-put assays described above can be followed (orsubstituted) by secondary screens in order to identify biologicalactivities which will, e.g., allow one skilled in the art todifferentiate agonists from antagonists. The type of a screen used willdepend on the desired activity that needs to be tested. For example, anassay can be developed in which the ability of a candidate agent tomodulate insulin secretion (e.g., from a β cell, islet tissue orpancreatic tissue) can be used to identify antagonists or agonists froma group of peptide fragments isolated though one of the primary screensdescribed above.

Peptide Mimetics

The invention also provides for production of the protein bindingdomains of components of the MCH signaling pathway, e.g., MCH or MCH-R,to generate mimetics, e.g. peptide or non-peptide agents, e.g.,inhibitory agents. See, for example, “Peptide inhibitors of humanpapillomavirus protein binding to retinoblastoma gene protein” Europeanpatent applications EP 0412 762and EP 0031 080.

Non-hydrolyzable peptide analogs of critical residues can be generatedusing benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistryand Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988), azepine (e.g., see Huffinan et al. in Peptides: Chemistry andBiology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988),substituted gama lactam rings (Garvey et al. in Peptides: Chemistry andBiology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988), keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem29:295; and Ewenson et al. in Peptides: Structure and Function(Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co.Rockland, Ill. 1985), b-turn dipeptide cores (Nagai et al. (1985)Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc Perkin Trans1:1231), and b-aminoalcohols (Gordon et al. (1985) Biochem Biophys ResCommunl26:419; and Dann et al. (1986) Biochem Biophys Res Commun134:71).

Antibodies

An agent described herein, e.g., a modulator of a component of the MCHsignaling pathway, e.g., MCH or MCH-R, can also be an antibodyspecifically reactive with a component of the MCH signaling pathway,e.g., MCH or MCH-R. An antibody can be an antibody or a fragmentthereof, e.g., an antigen binding portion thereof. As used herein, theterm “antibody” refers to a protein comprising at least one, andpreferably two, heavy (H) chain variable regions (abbreviated herein asVH), and at least one and preferably two light (L) chain variableregions (abbreviated herein as VL). The VH and VL regions can be furthersubdivided into regions of hypervariability, termed “complementaritydetermining regions” (“CDR”), interspersed with regions that are moreconserved, termed “framework regions” (FR). The extent of the frameworkregion and CDR's has been precisely defined (see, Kabat, E. A., et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, whichare incorporated herein by reference). Each VH and VL is composed ofthree CDR's and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The antibody can further include a heavy and light chain constantregion, to thereby form a heavy and light immunoglobulin chain,respectively. In one embodiment, the antibody is a tetramer of two heavyimmunoglobulin chains and two light immunoglobulin chains, wherein theheavy and light immunoglobulin chains are inter-connected by, e.g.,disulfide bonds. The heavy chain constant region is comprised of threedomains, CH1, CH2 and CH3. The light chain constant region is comprisedof one domain, CL. The variable region of the heavy and light chainscontains a binding domain that interacts with an antigen. The constantregions of the antibodies typically mediate the binding of the antibodyto host tissues or factors, including various cells of the immune system(e.g., effector cells) and the first component (Clq) of the classicalcomplement system.

The term “antigen-binding fragment” of an antibody (or simply “antibodyportion,” or “fragment”), as used herein, refers to one or morefragments of a full-length antibody that retain the ability tospecifically bind to an antigen (e.g., a polypeptide encoded by anucleic acid of Group I or II). Examples of binding fragmentsencompassed within the term “antigen-binding fragment” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of the VL,VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate nucleic acids, theycan be joined, using recombinant methods, by a synthetic linker thatenables them to be made as a single protein chain in which the VL and VHregions pair to form monovalent molecules (known as single chain Fv(scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chainantibodies are also intended to be encompassed within the term“antigen-binding fragment” of an antibody. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies. The term “monoclonal antibody” or “monoclonalantibody composition”, as used herein, refers to a population ofantibody molecules that contain only one species of an antigen bindingsite capable of immunoreacting with a particular epitope. A monoclonalantibody composition thus typically displays a single binding affinityfor a particular protein with which it immunoreacts.

Anti-protein/anti-peptide antisera or monoclonal antibodies can be madeas described herein by using standard protocols (See, for example,Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold SpringHarbor Press: 1988)).

A component of the MCH signaling pathway, e.g., MCH or MCH-R, can beused as an immunogen to generate antibodies that bind the componentusing standard techniques for polyclonal and monoclonal antibodypreparation. The full-length component protein can be used or,alternatively, antigenic peptide fragments of the component can be usedas immunogens.

Typically, a peptide is used to prepare antibodies by immunizing asuitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, a recombinant MCH or MCH-R peptide, or a chemically synthesizedMCH or MCH-R peptide or antagonist. See, e.g., U.S. Pat. No. 5,460,959;and co-pending U.S. applications Ser No. 08/334,797; U.S. Ser No.08/231,439; U.S. Ser No. 08/334,455; and U.S. Ser No. 08/928,881, whichare hereby expressly incorporated by, reference in their entirety. Thenucleotide and amino acid sequences of MCH and MCH-R described hereinare known. The preparation can further include an adjuvant, such asFreund's complete or incomplete adjuvant, or similar immunostimulatoryagent. Immunization of a suitable subject with an immunogenic componentof the MCH signaling pathway, e.g., MCH or MCH-R, or fragmentpreparation induces a polyclonal antibody response.

Additionally, antibodies produced by genetic engineering methods, suchas chimeric and humanized monoclonal antibodies, comprising both humanand non-human portions, which can be made using standard recombinant DNAtechniques, can be used. Such chimeric and humanized monoclonalantibodies can be produced by genetic engineering using standard DNAtechniques known in the art, for example using methods described inRobinson et al. International Application No. PCT/US86/02269; Akira, etal. European Patent Application 184,187; Taniguchi, M., European PatentApplication 171,496; Morrison et al. European Patent Application173,494; Neuberger et al. PCT International Publication No. WO 86/01533;Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European PatentApplication 125,023; Better et al., Science 240:1041-1043, 1988; Liu etal., PNAS 84:3439-3443, 1987; Liu et al., J. Immunol. 139:3521-3526,1987; Sun et al. PNAS 84:214-218, 1987; Nishimura et al., Canc. Res.47:999-1005, 1987; Wood et al., Nature 314:446-449, 1985; and Shaw etal., J. Natl. Cancer Inst. 80:1553-1559, 1988); Morrison, S. L., Science229:1202-1207, 1985; Oi et al., BioTechniques 4:214, 1986; Winter U.S.Pat. No. 5,225,539; Jones et al., Nature 321:552-525, 1986; Verhoeyan etal., Science 239:1534, 1988; and Beidler et al., J. Immunol.141:4053-4060, 1988.

In addition, a human monoclonal antibody directed against a component ofthe MCH signaling pathway, e.g., MCH or MCH-R, can be made usingstandard techniques. For example, human monoclonal antibodies can begenerated in transgenic mice or in immune deficient mice engrafted withantibody-producing human cells. Methods of generating such mice aredescribe, for example, in Wood et al. PCT publication WO 91/00906,Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. PCTpublication WO 92/03918; Kay et al. PCT publication WO 92/03917; Kay etal. PCT publication WO 93/12227; Kay et al. PCT publication 94/25585;Rajewsky et al. Pct publication WO 94/04667; Ditullio et al. PCTpublication WO 95/17085; Lonberg, N. et al. (1994) Nature 368:856-859;Green, L. L. et al. (1994) Nature Genet. 7:13-21; Morrison, S. L. et al.(1994) Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. (1993)Year Immunol 7:33-40; Choi et al. (1993) Nature Genet. 4:117-123;Tuaillon et al. (1993) PNAS 90:3720-3724; Bruggeman et al. (1991) Eur JImmunol 21:1323-1326); Duchosal et al. PCT publication WO 93/05796; U.S.Pat. No. 5,411,749; McCune et al. (1988) Science 241:1632-1639),Kamel-Reid et al. (1988) Science 242:1706; Spanopoulou (1994) Genes &Development 8:1030-1042; Shinkai et al. (1992) Cell 68:855-868). A humanantibody-transgenic mouse or an immune deficient mouse engrafted withhuman antibody-producing cells or tissue can be immunized with acomponent of the MCH signaling pathway, e.g., MCH or MCH-R, or anantigenic peptide thereof, and splenocytes from these immunized mice canthen be used to create hybridomas. Methods of hybridoma production arewell known.

Human monoclonal antibodies against a component of the MCH signalingpathway, e.g., MCH or MCH-R, can also be prepared by constructing acombinatorial immunoglobulin library, such as a Fab phage displaylibrary or a scFv phage display library, using immunoglobulin lightchain and heavy chain cDNAs prepared from mRNA derived from lymphocytesof a subject. See, e.g., McCafferty et al. PCT publication WO 92/01047;Marks et al. (1991) J. Mol. Biol. 222:581-597; and Griffths et al.(1993) EMBO J 12:725-734. In addition, a combinatorial library ofantibody variable regions can be generated by mutating a known humanantibody. For example, a variable region of a human antibody known tobind a component of the MCH signaling pathway, e.g., MCH or MCH-R, canbe mutated, by for example using randomly altered mutagenizedoligonucleotides, to generate a library of mutated variable regionswhich can then be screened to bind to a component of the MCH signalingpathway, e.g., MCH or MCH-R. Methods of inducing random mutagenesiswithin the CDR regions of immunoglobin heavy and/or light chains,methods of crossing randomized heavy and light chains to form pairingsand screening methods can be found in, for example, Barbas et al. PCTpublication WO 96/07754; Barbas et al. (1992) Proc. Nat'l Acad. Sci. USA89:4457-4461.

The immunoglobulin library can be expressed by a population of displaypackages, preferably derived from filamentous phage, to form an antibodydisplay library. Examples of methods and reagents particularly amenablefor use in generating antibody display library can be found in, forexample, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTpublication WO 92/18619; Dower et al. PCT publication WO 91/17271;Winter et al. PCT publication WO 92/20791; Markland et al. PCTpublication WO 92/15679; Breitling et al. PCT publication WO 93/01288;McCafferty et al. PCT publication WO 92/01047; Garrard et al. PCTpublication WO 92/09690; Ladner et al. PCT publication WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) HumAntibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffths et al. (1993) supra; Hawkins et al. (1992) J Mol Biol226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) PNAS 88:7978-7982. Once displayed on the surface ofa display package (e.g., filamentous phage), the antibody library isscreened to identify and isolate packages that express an antibody thatbinds a component of the MCH signaling pathway, e.g., MCH or MCH-R. In apreferred embodiment, the primary screening of the library involvespanning with an immobilized component of the MCH signaling pathway,e.g., MCH or MCH-R, and display packages expressing antibodies that bindimmobilized proteins described herein are selected.

Antisense Nucleic Acid Sequences

Nucleic acid molecules which are antisense to a nucleotide encoding acomponent of the MCH signaling pathway, e.g., MCH or MCH-R, can also beused as an agent which inhibits expression of MCH signaling. An“antisense” nucleic acid includes a nucleotide sequence which iscomplementary to a “sense” nucleic acid encoding the component, e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. Accordingly, an antisense nucleicacid can form hydrogen bonds with a sense nucleic acid. The antisensenucleic acid can be complementary to an entire coding strand, or to onlya portion thereof. For example, an antisense nucleic acid molecule whichantisense to the “coding region” of the coding strand of a nucleotidesequence encoding the component can be used.

The coding strand sequences encoding MCH and MCH-R are known. Given thecoding strand sequences encoding these proteins, antisense nucleic acidscan be designed according to the rules of Watson and Crick base pairing.The antisense nucleic acid molecule can be complementary to the entirecoding region of mRNA, but more preferably is an oligonucleotide whichis antisense to only a portion of the coding or noncoding region ofmRNA. For example, the antisense oligonucleotide can be complementary tothe region surrounding the translation start site of the mRNA. Anantisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acidcan be constructed using chemical synthesis and enzymatic ligationreactions using procedures known in the art. For example, an antisensenucleic acid (e.g., an antisense oligonucleotide) can be chemicallysynthesized using naturally occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleic acids, e.g., phosphorothioatederivatives and acridine substituted nucleotides can be used. Examplesof modified nucleotides which can be used to generate the antisensenucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest.

Administration

An agent that modulates a component of the MCH signaling pathway, e.g.,MCH or MCH-R, e.g., an agent described herein, can be administered to asubject by standard methods. For example, the agent can be administeredby any of a number of different routes including intravenous,intradermal, subcutaneous, oral (e.g., inhalation), transdermal(topical), and transmucosal, or direct administration, e.g., onto thesurface of the eye. In one embodiment, the modulating agent can beadministered orally. In another embodiment, the agent is administered byinjection, e.g., intramuscularly, or intravenously. In a preferredembodiment, the agent is administered directly onto the surface of theeye.

The agent that modulates a component of the MCH signaling pathway, e.g.,MCH or MCH-R, e.g., an agent described herein, e.g., nucleic acidmolecules, polypeptides, fragments or analogs, modulators, organiccompounds and antibodies (also referred to herein as “active compounds”)can be incorporated into pharmaceutical compositions suitable foradministration to a subject, e.g., a human. Such compositions typicallyinclude the nucleic acid molecule, polypeptide, modulator, or antibodyand a pharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances are known. Except insofaras any conventional media or agent is incompatible with the activecompound, such media can be used in the compositions of the invention.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition can be formulated to be compatible with itsintended route of administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., an agent described herein) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active compounds are formulated into ointments, salves, gels, orcreams as generally known in the art.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

The nucleic acid molecules described herein can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al., PNAS 91:3054-3057, 1994). Thepharmaceutical preparation of the gene therapy vector can include thegene therapy vector in an acceptable diluent, or can include a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Gene Therapy

The nucleic acids described herein, e.g., an antisense nucleic aciddescribed herein, can be incorporated into gene constructs to be used asa part of a gene therapy protocol to deliver nucleic acids encodingeither an agonistic or antagonistic form of a molecule described herein.The invention features expression vectors for in vivo transfection andexpression of an MCH signaling molecule described herein in particularcell types so as to reconstitute the function of, or alternatively,antagonize the function of the component in a cell in which thatpolypeptide is misexpressed. Expression constructs of such componentsmay be administered in any biologically effective carrier, e.g. anyformulation or composition capable of effectively delivering thecomponent gene to cells in vivo. Approaches include insertion of thesubject gene in viral vectors including recombinant retroviruses,adenovirus, adeno-associated virus, and herpes simplex virus-1, orrecombinant bacterial or eukaryotic plasmids. Viral vectors transfectcells directly; plasmid DNA can be delivered with the help of, forexample, cationic liposomes (lipofectin) or derivatized (e.g. antibodyconjugated), polylysine conjugates, gramacidin S, artificial viralenvelopes or other such intracellular carriers, as well as directinjection of the gene construct or CaPO4 precipitation carried out invivo.

A preferred approach for in vivo introduction of nucleic acid into acell is by use of a viral vector containing nucleic acid, e.g. a cDNA,encoding a component of the MCH signaling pathway, e.g., MCH or MCH-R.Infection of cells with a viral vector has the advantage that a largeproportion of the targeted cells can receive the nucleic acid.Additionally, molecules encoded within the viral vector, e.g., by a cDNAcontained in the viral vector, are expressed efficiently in cells whichhave taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors can be used as arecombinant gene delivery system for the transfer of exogenous genes invivo, particularly into humans. These. vectors provide efficientdelivery of genes into cells, and the transferred nucleic acids arestably integrated into the chromosomal DNA of the host. The developmentof specialized cell lines (termed “packaging cells”) which produce onlyreplication-defective retroviruses has increased the utility ofretroviruses for gene therapy, and defective retroviruses arecharacterized for use in gene transfer for gene therapy purposes (for areview see Miller, A. D. (1990) Blood 76:271). A replication defectiveretrovirus can be packaged into virions which can be used to infect atarget cell through the use of a helper virus by standard techniques.Protocols for producing recombinant retroviruses and for infecting cellsin vitro or in vivo with such viruses can be found in Current Protocolsin Molecular Biology, Ausubel, F. M. et al. (eds.) Greene PublishingAssociates, (1989), Sections 9.10-9.14 and other standard laboratorymanuals. Examples of suitable retroviruses include pLJ, pZIP, pWE andpEM which are known to those skilled in the art. Examples of suitablepackaging virus lines for preparing both ecotropic and amphotropicretroviral systems include *Crip, *Cre, *2 and *Am. Retroviruses havebeen used to introduce a variety of genes into many different celltypes, including epithelial cells, in vitro and/or in vivo (see forexample Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan(1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988)Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc.Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad.Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

Another viral gene delivery system useful in the present inventionutilizes adenovirus-derived vectors. The genome of an adenovirus can bemanipulated such that it encodes and expresses a gene product ofinterest but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See, for example, Berkner et al. (1988)BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; andRosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 dl324 or other strains ofadenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known to those skilled in theart. Recombinant adenoviruses can be advantageous in certaincircumstances in that they are not capable of infecting nondividingcells and can be used to infect a wide variety of cell types, includingepithelial cells (Rosenfeld et al. (1992) cited supra). Furthermore, thevirus particle is relatively stable and amenable to purification andconcentration, and as above, can be modified so as to affect thespectrum of infectivity. Additionally, introduced adenoviral DNA (andforeign DNA contained therein) is not integrated into the genome of ahost cell but remains episomal, thereby avoiding potential problems thatcan occur as a result of insertional mutagenesis in situ whereintroduced DNA becomes integrated into the host genome (e.g., retroviralDNA). Moreover, the carrying capacity of the adenoviral genome forforeign DNA is large (up to 8 kilobases) relative to other gene deliveryvectors (Berkner et al. cited supra; Haj-Ahmand and Graham (1986) J.Virol. 57:267).

Yet another viral vector system useful for delivery of the subject geneis the adeno-associated virus (AAV). Adeno-associated virus is anaturally occurring defective virus that requires another virus, such asan adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka etal. (1992) Curr. Topics in Micro. and Immunol.158:97-129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356;Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al.(1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of acomponent of the MCH signaling pathway, e.g., MCH or MCH-R, in thetissue of a subject. Most nonviral methods of gene transfer rely onnormal mechanisms used by mammalian cells for the uptake andintracellular transport of macromolecules. In preferred embodiments,non-viral gene delivery systems of the present invention rely onendocytic pathways for the uptake of the subject gene by the targetedcell. Exemplary gene delivery systems of this type include liposomalderived systems, poly-lysine conjugates, and artificial viral envelopes.Other embodiments include plasmid injection systems such as aredescribed in Meuli et al. (2001) J Invest Dermatol. 116(1):131-135;Cohen et al. (2000) Gene Ther 7(22):1896-905; or Tam et al. (2000) GeneTher 7(21):1867-74.

In a representative embodiment, a gene encoding a component of the MCHsignaling pathway, e.g., MCH or MCH-R, can be entrapped in liposomesbearing positive charges on their surface (e.g., lipofectins) and(optionally) which are tagged with antibodies against cell surfaceantigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka20:547-551; PCT publication WO 91/06309; Japanese patent application1047381; and European patent publication EP-A-43075).

In clinical settings, the gene delivery systems for the therapeutic genecan be introduced into a patient by any of a number of methods, each ofwhich is familiar in the art. For instance, a pharmaceutical preparationof the gene delivery system can be introduced systemically, e.g. byintravenous injection, and specific transduction of the protein in thetarget cells occurs predominantly from specificity of transfectionprovided by the gene delivery vehicle, cell-type or tissue-typeexpression due to the transcriptional regulatory sequences controllingexpression of the receptor gene, or a combination thereof. In otherembodiments, initial delivery of the recombinant gene is more limitedwith introduction into the animal being quite localized. For example,the gene delivery vehicle can be introduced by catheter (see U.S. Pat.No. 5,328,470) or by stereotactic injection (e.g. Chen et al. (1994)PNAS 91: 3054-3057).

The pharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery system can beproduced in tact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system.

Cell Therapy

A component of the MCH signaling pathway, e.g., MCH or MCH-R, can alsobe increased in a subject by introducing into a cell, e.g., anendothelial cell, a nucleotide sequence that modulates the production ofa component of the MCH signaling pathway, e.g., MCH or MCH-R, e.g., anucleotide sequence encoding MCH or MCH-R polypeptide or functionalfragment or analog thereof, a promoter sequence, e.g., a promotersequence from an MCH or MCH-R gene or from another gene; an enhancersequence, e.g., 5′ untranslated region (UTR), e.g., a 5′ UTR, a 3′ UTR;a polyadenylation site; an insulator sequence; or another sequence thatmodulates the expression of a component of the MCH signaling pathway,e.g., MCH or MCH-R. The cell can then be introduced into the subject.

Primary and secondary cells to be genetically engineered can be obtainedform a variety of tissues and include cell types which can be maintainedpropagated in culture. For example, primary and secondary cells includefibroblasts, keratinocytes, epithelial cells (e.g., mammary epithelialcells, intestinal epithelial cells), endothelial cells, glial cells,neural cells, formed elements of the blood (e.g., lymphocytes, bonemarrow cells), muscle cells (myoblasts) and precursors of these somaticcell types. Primary cells are preferably obtained from the individual towhom the genetically engineered primary or secondary cells areadministered. However, primary cells may be obtained for a donor (otherthan the recipient).

The term “primary cell” includes cells present in a suspension of cellsisolated from a vertebrate tissue source (prior to their being platedi.e., attached to a tissue culture substrate such as a dish or flask),cells present in an explant derived from tissue, both of the previoustypes of cells plated for the first time, and cell suspensions derivedfrom these plated cells. The term “secondary cell” or “cell strain”refers to cells at all subsequent steps in culturing. Secondary cellsare cell strains which consist of secondary cells which have beenpassaged one or more times.

Primary or secondary cells of vertebrate, particularly mammalian, origincan be transfected with an exogenous nucleic acid sequence whichincludes a nucleic acid sequence encoding a signal peptide, and/or aheterologous nucleic acid sequence, e.g., encoding a component of theMCH signaling pathway, e.g., MCH or MCH-R, or an agonist or antagonistthereof, and produce the encoded product stably and reproducibly invitro and in vivo, over extended periods of time. A heterologous aminoacid can also be a regulatory sequence, e.g., a promoter, which causesexpression, e.g., inducible expression or upregulation, of an endogenoussequence. An exogenous nucleic acid sequence can be introduced into aprimary or secondary cell by homologous recombination as described, forexample, in U.S. Pat. No.: 5,641,670, the contents of which areincorporated herein by reference. The transfected primary or secondarycells may also include DNA encoding a selectable marker which confers aselectable phenotype upon them, facilitating their identification andisolation.

Vertebrate tissue can be obtained by standard methods such a punchbiopsy or other surgical methods of obtaining a tissue source of theprimary cell type of interest. For example, punch biopsy is used toobtain skin as a source of fibroblasts or keratinocytes. A mixture ofprimary cells is obtained from the tissue, using known methods, such asenzymatic digestion or explanting. If enzymatic digestion is used,enzymes such as collagenase, hyaluronidase, dispase, pronase, trypsin,elastase and chymotrypsin can be used.

The resulting primary cell mixture can be transfected directly or it canbe cultured first, removed from the culture plate and resuspended beforetransfection is carried out. Primary cells or secondary cells arecombined with exogenous nucleic acid sequence to, e.g., stably integrateinto their genomes, and treated in order to accomplish transfection. Asused herein, the term “transfection” includes a variety of techniquesfor introducing an exogenous nucleic acid into a cell including calciumphosphate or calcium chloride precipitation, microinjection,DEAE-dextrin-mediated transfection, lipofection or electrophoration, allof which are routine in the art.

Transfected primary or secondary cells undergo sufficient numberdoubling to produce either a clonal cell strain or a heterogeneous cellstrain of sufficient size to provide the therapeutic protein to anindividual in effective amounts. The number of required cells in atransfected clonal heterogeneous cell strain is variable and depends ona variety of factors, including but not limited to, the use of thetransfected cells, the fuictional level of the exogenous DNA in thetransfected cells, the site of implantation of the transfected cells(for example, the number of cells that can be used is limited by theanatomical site of implantation), and the age, surface area, andclinical condition of the patient.

The transfected cells, e.g., cells produced as described herein, can beintroduced into an individual to whom the product is to be delivered.Various routes of administration and various sites (e.g., renal subcapsular, subcutaneous, central nervous system (including intrathecal),intravascular, intrahepatic, intrasplanchnic, intraperitoneal (includingintraomental), intramuscularly implantation) can be used. One implantedin individual, the transfected cells produce the product encoded by theheterologous DNA or are affected by the heterologous DNA itself. Forexample, an individual who suffers from an antibody-mediated arthriticdisorder is a candidate for implantation of cells producing anantagonist of a component of the MCH signaling pathway, e.g., MCH orMCH-R.

An immunosuppressive agent e.g., drug, or antibody, can be administeredto a subject at a dosage sufficient to achieve the desired therapeuticeffect (e.g., inhibition of rejection of the cells). Dosage ranges forimmunosuppressive drugs are known in the art. See, e.g., Freed et al.(1992) N. Engl. J. Med. 327:1549; Spencer et al. (1992) N. Engl. J. Med.327:1541′ Widner et al. (1992) n. Engl. J. Med. 327:1556). Dosage valuesmay vary according to factors such as the disease state, age, sex, andweight of the individual.

Diagnostic Assays

The diagnostic assays described herein involve evaluating level,expression, or activity of a component of the MCH signaling pathway,e.g., MCH or MCH-R, e.g., an MCH-R described herein. Protein levels canbe quantitated in a variety of ways well known in the art, such asimmunoprecipitation, Western blot analysis (immunoblotting), ELISA orfluorescence-activated cell sorting (FACS). Also, various art-recognizedmethods are known and/or are commercially available for evaluatinginsulin secretion from, e.g., a β cell, islet, pancreatic tissue oranimal, e.g., a human.

Another method of evaluating MCH signaling in a subject is to determinethe presence or absence of a lesion in, or the misexpression of, a genethat encodes a component of the MCH signaling pathway, e.g., MCH orMCH-R, e.g., an MCH-R described herein. The method includes one or moreof the following:

detecting, in a tissue of the subject, the presence or absence of amutation which affects the expression of a gene encoding a component ofthe MCH signaling pathway, e.g., MCH or MCH-R, or detecting the presenceor absence of a mutation in a region which controls the expression ofthe gene, e.g., a mutation in the 5′ control region;

detecting, in a tissue of the subject, the presence or absence of amutation which alters the structure of a gene encoding a component ofthe MCH signaling pathway, e.g., MCH or MCH-R;

detecting, in a tissue of the subject, the misexpression of a geneencoding a component of the MCH signaling pathway, e.g., MCH or MCH-R,at the mRNA level, e.g., detecting a non-wild type level of a mRNA;

detecting, in a tissue of the subject, the misexpression of the gene, atthe protein level, e.g., detecting a non-wild type level of a componentof the MCH signaling pathway, e.g., MCH or MCH-R polypeptide.

In preferred embodiments the method includes: ascertaining the existenceof at least one of: a deletion of one or more nucleotides from a geneencoding a component of the MCH signaling pathway, e.g., MCH or MCH-R;an insertion of one or more nucleotides into the gene, a point mutation,e.g., a substitution of one or more nucleotides of the gene, a grosschromosomal rearrangement of the gene, e.g., a translocation, inversion,or deletion.

For example, detecting the genetic lesion can include: (i) providing aprobe/primer including an oligonucleotide containing a region ofnucleotide sequence which hybridizes to a sense or antisense sequencefrom a gene encoding a component of the MCH signaling pathway, e.g., MCHor MCH-R, or naturally occurring mutants thereof or 5′ or 3′ flankingsequences naturally associated with the gene; (ii) exposing theprobe/primer to nucleic acid of a tissue; and detecting, byhybridization, e.g., in situ hybridization, of the probe/primer to thenucleic acid, the presence or absence of the genetic lesion.

In preferred embodiments detecting the misexpression includesascertaining the existence of at least one of: an alteration in thelevel of a messenger RNA transcript of a gene encoding a component ofthe MCH signaling pathway, e.g., MCH or MCH-R; the presence of anon-wild type splicing pattern of a messenger RNA transcript of thegene; or a non-wild type level of a gene encoding a component of the MCHsignaling pathway, e.g., MCH or MCH-R.

In some embodiments, the method includes determining the structure of agene encoding a component of the MCH signaling pathway, e.g., MCH orMCH-R, an abnormal structure being indicative of risk for the disorder.In other embodiments, the method includes contacting a sample from thesubject with an antibody to a component of the MCH signaling pathway,e.g., MCH or MCH-R, or a nucleic acid which hybridizes specifically withthe gene encoding the component of the MCH signaling pathway, e.g., MCHor MCH-R.

Expression Monitoring and Profiling.

The presence, level, or absence of a component of the MCH signalingpathway, e.g., MCH or MCH-R (protein or nucleic acid) in a biologicalsample can be evaluated by obtaining a biological sample from a testsubject and contacting the biological sample with a compound or an agentcapable of detecting the protein or nucleic acid (e.g., mRNA, genomicDNA) that encodes a component of the MCH signaling pathway, e.g., MCH orMCH-R, such that the presence of the protein or nucleic acid is detectedin the biological sample. The term “biological sample” includes tissues,cells and biological fluids isolated from a subject, as well as tissues,cells and fluids present within a subject, e.g., synovial fluid.Preferred biological samples are serum or synovial fluid. The level ofexpression of the component of the MCH signaling pathway, e.g., MCH orMCH-R, can be measured in a number of ways, including, but not limitedto: measuring the mRNA encoded by the gene; measuring the amount ofprotein encoded by the gene of; or measuring the activity of the proteinencoded by the gene.

The level of mRNA corresponding to a gene encoding a component of theMCH signaling pathway, e.g., MCH or MCH-R, in a cell can be determinedboth by in situ and by in vitro formats.

Isolated mRNA can be used in hybridization or amplification assays thatinclude, but are not limited to, Southern or Northern analyses,polymerase chain reaction analyses and probe arrays. One preferreddiagnostic method for the detection of mRNA levels involves contactingthe isolated mRNA with a nucleic acid molecule (probe) that canhybridize to the mRNA encoded by the gene being detected. The nucleicacid probe can be, for example, a full-length nucleic acid, or a portionthereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250or 500 nucleotides in length and sufficient to specifically hybridizeunder stringent conditions to mRNA or genomic DNA of a component of theMCH signaling pathway, e.g., MCH or MCH-R. The probe can be disposed onan address of an array, e.g., an array described below. Other suitableprobes for use in the diagnostic assays are described herein.

In one format, mRNA (or cDNA) is immobilized on a surface and contactedwith the probes, for example by running the isolated mRNA on an agarosegel and transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probes are immobilized ona surface and the mRNA (or cDNA) is contacted with the probes, forexample, in a two-dimensional gene chip array described below. A skilledartisan can adapt known mRNA detection methods for use in detecting thelevel of mRNA encoded by the gene of a component of the MCH signalingpathway, e.g., MCH or MCH-R.

The level of mRNA in a sample that is encoded by a gene can be evaluatedwith nucleic acid amplification, e.g., by rtPCR (Mullis (1987) U.S. Pat.No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad.Sci. USA 88:189-193), self sustained sequence replication (Guatelli etal., (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptionalamplification system (Kwoh et al., (1989), Proc. Natl. Acad. Sci. USA86:1173-1177), Q-Beta Replicase (Lizardi et al., (1988) Bio/Technology6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No.5,854,033) or any other nucleic acid amplification method, followed bythe detection of the amplified molecules using techniques known in theart. As used herein, amplification primers are defined as being a pairof nucleic acid molecules that can anneal to 5′ or 3′ regions of a gene(plus and minus strands, respectively, or vice-versa) and contain ashort region in between. In general, amplification primers are fromabout 10 to 30 nucleotides in length and flank a region from about 50 to200 nucleotides in length. Under appropriate conditions and withappropriate reagents, such primers permit the amplification of a nucleicacid molecule comprising the nucleotide sequence flanked by the primers.

For in situ methods, a cell or tissue sample can be prepared/processedand immobilized on a support, typically a glass slide, and thencontacted with a probe that can hybridize to mRNA that encodes the genebeing analyzed.

In another embodiment, the methods further contacting a control samplewith a compound or agent capable of detecting mRNA, or genomic DNA of acomponent of the MCH signaling pathway, e.g., MCH or MCH-R, andcomparing the presence of the mRNA or genomic DNA in. the control samplewith the presence of mRNA or genomic DNA of a component of the MCHsignaling pathway, e.g., MCH or MCH-R, in the test sample. In stillanother embodiment, serial analysis of gene expression, as described inU.S. Pat. No. 5,695,937, is used to detect transcript levels of acomponent of the MCH signaling pathway, e.g., MCH or MCH-R, describedherein.

A variety of methods can be used to determine the level of proteinencoded by a gene of a component of the MCH signaling pathway, e.g., MCHor MCH-R. In general, these methods include contacting an agent thatselectively binds to the protein, such as an antibody with a sample, toevaluate the level of protein in the sample. In a preferred embodiment,the antibody bears a detectable label. Antibodies can be polyclonal, ormore preferably, monoclonal. An intact antibody, or a fragment thereof(e.g., Fab or F(ab′)2) can be used. The term “labeled”, with regard tothe probe or antibody, is intended to encompass direct labeling of theprobe or antibody by coupling (i.e., physically linking) a detectablesubstance to the probe or antibody, as well as indirect labeling of theprobe or antibody by reactivity with a detectable substance. Examples ofdetectable substances are provided herein.

The detection methods can be used to detect a component of the MCHsignaling pathway, e.g., MCH or MCH-R in a biological sample in vitro aswell as in vivo. In vitro techniques for detection of a component of theMCH signaling pathway, e.g., MCH or MCH-R, include enzyme linkedimmunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence,enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blotanalysis. In vivo techniques for detection of a component of the MCHsignaling pathway, e.g., MCH or MCH-R, include introducing into asubject a labeled antibody. For example, the antibody can be labeledwith a radioactive marker whose presence and location in a subject canbe detected by standard imaging techniques. In another embodiment, thesample is labeled, e.g., biotinylated and then contacted to theantibody, e.g., an antibody positioned on an antibody array. The samplecan be detected, e.g., with avidin coupled to a fluorescent label.

In another embodiment, the methods further include contacting thecontrol sample with a compound or agent capable of detecting a componentof the MCH signaling pathway, e.g., MCH or MCH-R, and comparing thepresence of the component protein in the control sample with thepresence of the component protein in the test sample.

The invention also includes kits for detecting the presence of acomponent of the MCH signaling pathway, e.g., MCH or MCH-R, in abiological sample. For example, the kit can include a compound or agentcapable of detecting protein (e.g., an antibody) or mRNA (e.g., anucleic acid probe) of a component of the MCH signaling pathway, e.g.,MCH or MCH-R, in a biological sample; and a standard. The compound oragent can be packaged in a suitable container. The kit can furthercomprise instructions for using the kit to evaluate a subject, e.g., forrisk or predisposition to an ocular disorder, e.g., an ocular disorderdescribed herein.

The diagnostic methods described herein can identify subjects having, orat risk of developing, an insulin related disorder, e.g., an insulinrelated disorder described herein.

The prognostic assays described herein can be used to determine whethera subject can be administered an agent (e.g., an agent that modulates acomponent of the MCH signaling pathway, e.g., MCH or MCH-R, e.g., anagent described herein) to treat an insulin related disorder, e.g., aninsulin related disorder described herein.

All references cited in this application are incorporated herein byreference.

EXAMPLES Example 1 Production and Analysis of MCH Overexpressing Mice

Production of transgenic animals. Restriction analysis of one P₁ clonecontaining the murine MCH gene allowed for the creation of anunambiguous physical map showing the position and orientation of theMCH-coding region. The clone consists of the expected 16-kb P₁ vectorand a 72-kb genomic insert. The 70-kb DNA construct used for generationof transgenic mice included approximately 25 kb of 5′ and 45 kb of 3′sequence flanking the MCH-coding region.

Fifty-four offspring were obtained from injection of about 200 oocytes,five of which were found to be transgenic by Southern blot analysis. Acolony from one of these founders with the highest evident gene copynumber (approximately five, with one apparent integration site) wasestablished. The transgene has remained stably integrated and hasdemonstrated simple Mendelian inheritance. Both heterozygous andhomozygous transgenic animals appeared healthy and demonstrated no grossanatomic or behavioral abnormalities.

Overexpression of the MCH transgene. Northern blot analysis performed onhypothalamic tissue of heterozygous transgenic mice showed up to afourfold difference in MCH mRNA expression between wild-type littermatesand transgenic animals in the fed state (data not shown). The averagedifference between wild-type (n=9) and overexpresser mice (n=8) wasalmost twofold (45.1±3.0 versus 84.6±3.4 arbitrary units, P<0.0001 by ttest). MCH overexpression was confirmed by in situ hybridizationhistochemistry, which indicated a 50% increase in MCH expression in thefed state. The pattern of distribution of MCH message in the transgenicanimals is indistinguishable from that of wild-type mice, as assessed byin situ hybridization studies. No MCH signal could be detected byNorthern blot analysis in mRNA from various organs in the peripheryincluding liver, spleen, lung, heart, brown adipose tissue, and whiteadipose tissue. These data indicate that MCH expression is eutopic inMCH-OE. Immunohistochemical analysis of MCH levels indicated a visuallyevident increase in MCH immunoreactivity in MCH-OE mice compared withwild type mice.

Body weight, food intake, and percentage of body adiposity. Weight gainof heterozygous and homozygous transgenic mice was studied under severalconditions. Heterozygotes fed standard chow or a high-fat diet showed nodifferences in body weight compared with wild-type littermates.Homozygotes fed standard chow showed a nonsignificant tendency to beheavier than wild-type animals raised under identical conditions. Whenfed a high-fat diet, however, homozygotes gained significantly moreweight than wild-type mice, with a difference of 12.6% by age 13 weeks(P<0.001). The greater body weight of homozygous transgenic mice on ahigh-fat diet appears to be, at least in part, attributable to increasedfood intake, as these animals ate about 10% more than wild-typemice(P<0.001). Moreover, these mice were fatter than wild-type mice, asdemonstrated by elevated serum leptin concentration (25.6±1.9 vs.15.0±1.7 ng/ml; P<0.001) and carcass analysis (21.9±1.4 vs. 16.7±1.4%body fat; P=0.02).

Glucose homeostasis. Mean blood glucose determined at the end of thelight cycle (e.g., preprandial) for homozygous transgenic mice comparedwith wild-type mice was 181±4 versus 161±5 mg/dl (P=0.003),respectively. Transgenic mice also had higher mean blood glucosemeasured for 2 hours after intraperitoneal glucose injection thanwild-type mice (369±19 vs. 296±14 mg/dl; P=0.002). Mean plasma insulinconcentration determined at the beginning of the light cycle (e.g.,postprandial) was dramatically higher in the homozygous transgeniccompared with wild-type animals (9.5±1.7 vs. 1.0±0.2 ng/ml; P<0.001).Fifteen minutes after injection of insulin, mean blood glucose decreasedby only 5% in the homozygous transgenics compared with 31% in thewild-type mice (difference between groups for blood glucose response:P<0.001). Finally, pancreatic islet histology of transgenic micedemonstrated marked increase in islet size.

Example 2 MCH Stimulation Of Insulin Secretion

MCH stimulation of overnight cultured islets isolated from C57B1/6J miceshowed a concentration- and time-dependent insulin secretion. A 2.4-foldstimulation was observed at 100 nM after 30 min incubation in thepresence of 11.1 mM glucose (Control 0.86±0.22 vs MCH 2.1±0.11% ofinsulin content, n=3, p<0.02). A similar stimulatory effect was evidentwhen a mouse clonal β-cell line (βTC3) was treated with MCH, showing adirect effect of the peptide on the β-cells. MCH treatment of isletsisolated from MCH over expressing mice showed a 4-fold stimulatoryresponse (Control 2.8±0.36 vs 11.4±0.49 pg/μg protein, n=2) showing anenhanced effect of MCH in the presence of hyperinsulinemia.

Example 3 Expression of MCH and MCH Receptor in Islets

Using immunohistochemistry, it has been found that MCH is present in theislet.

To evaluate whether MCH stimulated insulin secretion via its receptor,RNA was prepared from mouse islets and the clonal β-cells and MCHreceptor expression levels were examined using Taqman analysis.Independent experiments showed the presence of MCH receptor (MCHR1)expression in the islets and in the clonal β-cells using thehypothalamus as a positive control. MCH receptor expression levels were9-fold lower in the islets (Hypothalamus 919.0 vs Islet 100.0 arbitraryunits) and 12-fold lower in the β-cell lines (Hypothalamus 7771.0 vsβ-cells 633.5 arbitrary units) compared to the levels in thehypothalamus. These data indicate that MCH stimulates insulin releasefrom the islets/β-cells via its own receptor.

To begin to examine the mechanism of action of MCH in the islets theeffect of the peptide on MAP kinase activity was investigated. Noconsistent effect could be detected in MAP kinase levels in islets orclonal β-cells treated with 1 μM MCH indicating a MAPK-independenteffect.

Taken together these data suggest that MCH directly stimulates insulinsecretion and provides a novel system to study MCH signaling pathways.

1. A method of promoting β cell function or development, selected fromthe group consisting of increasing insulin secretion, β cell size, and βbeta cell growth the method comprising contacting a β cell with aneffective amount of an agonist of melanocyte concentrating hormone(MCH), wherein the agonist comprises MCH or a peptide analog of MCHcomprising the sequence VYRPC (amino acids 12-16 of SEQ ID NO:1),wherein the agonist has MCH activity in a fish skin teleost bioassay. 2.The method of claim 1, wherein the agonist is contacted with the β cellin vitro.
 3. The method of claim 2, further comprising implanting thecell into a subject.
 4. The method of claim 3, wherein the subject is aliving mammal.
 5. The method of claim 4, wherein the mammal is a human.6. The method of claim 4, wherein the mammal is at risk for or has aninsulin related disorder.
 7. The method of claim 3, wherein the subjectis a non-human animal.
 8. The method of claim 7, wherein the animal isan animal model of an insulin related disorder.
 9. The method of claim2, wherein the β cell is in an isolated pancreatic tissue.
 10. Themethod of claim 9, wherein the isolated pancreatic tissue comprises anislet or islet equivalent.
 11. The method of claim 2, wherein the β cellis autologous to the subject.
 12. The method of claim 2, wherein theagonist is contacted with the β cell in vivo.
 13. The method of claim 2,wherein the β cell is in a living mammal.
 14. The method of claim 13,wherein the mammal is a human.
 15. The method of claim 12, wherein themammal is at risk for or has an insulin related disorder.
 16. The methodof claim 12, wherein the β cell is in a non-human animal.
 17. The methodof claim 16, wherein the animal is an animal model of an insulin relateddisorder.